Journal of Immune Based Therapies, Vaccines and Antimicrobials, 2013, 2, 15-27
http://dx.doi.org/10.4236/jibtva.2013.22003 Published Online April 2013 (http://www.scirp.org/journal/jibtva)
Towards a Pan-Anti-Allergy Vaccine
Sari S. Sabban1*#, Hongtu Ye2*, Athanassios Vratimos3*, Arthur J. G. Moir2,
Alan W. Wheeler4, Birgit A. Helm2
1Dr. Soliman Fakeeh College of Nursing and Medical Sciences, Jeddah, KSA
2The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology,
The University of Sheffield, Sheffield, UK
3Molecular Diagnostics Laboratory, National Centre for Scientific Research “Demokritos”, Athens, Greece
4Allergy Therapeutics Ltd., Worthing, UK
Received April 30, 2013; revised May 24, 2013; accepted June 5, 2013
Copyright © 2013 Sari S. Sabban et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Allergic manifestations affect 20% - 30% of the population in industrialized countries. The global market for asthma
and allergy medications has been estimated to exceed USD 6 billion, since 40% of the human population has some form
of IgE sensitization to diverse proteins. Most therapeutic intervention strategies cope with the symptoms of allergy
without eliminating the underlying cause and many are associated with undesirable and often long-term debilitating side
effects. We designed a peptide immunogen encompassing sequences of the human Cε2-3 linker region to prime rat
(Rattus norvegicus) immune systems, we then designed a chimeric human-dog-human IgE antibody and used it to boost
the immune system and produce high-affinity antibodies that targets native IgE. The investigation showed that this pep-
tide immunogen elicit the formation of antibodies recognizing the native IgE of human, canine and equine origin. The
current investigation describes novel approaches aimed at the development of safe anti-allergy vaccines based on active
immunization with IgE-derived peptides that are involved in the complementary interaction with the high affinity re-
ceptor. The immunization strategy was successful but did not fully work as predicted, thus we propose that peptides
described in the current study may lead to the development of a pan-anti-allergy vaccine with applications for the
treatment of all IgE-mediated allergic response independent of the nature of the offending allergen.
Keywords: Anti-Allergy Vaccine; Hypersensitivity Responses; IgE; Immunotherapy; Original Antigenic Sin
1. Introduction
IgE antibodies are best known for their role as mediators
of the allergic response, which in its most serious mani-
festations causes asthma and anaphylactic shock, re-
viewed in [1]. The current consensus is that allergic dis-
eases become manifest as a result of an imbalance be-
tween Th1 and Th2 responses to environmental antigens/
pollutants capable of creating a cytokine environment
favoring Th2 immune response. IgE mediates allergic
responses by sensitizing cells expressing high- and low-
affinity receptors for allergen-induced release of phar-
macologically potent chemicals causing the symptoms
associated with the diverse manifestations of the disease.
Severity of symptoms ranges from mild to high and can
be life threatening.
The high socio-economic cost of management of al-
lergic disorders initiated the quest for effective therapeu-
tic intervention strategies which started with the demon-
stration that a proteolytic fragment derived from human
IgE inhibited the sensitization of mast cells with aller-
gen-specific IgE [2], reviewed in [3]. Subsequently, pro-
gressively smaller IgE, and receptor (FcεRIα), derived
peptides that can competitively inhibit ligand/receptor
interaction, were developed [4], but the low affinity of
these peptidomimetics proved a major drawback under-
lying this strategy. Furthermore, peptides based on re-
ceptor-derived sequences carry a potential danger of
stimulating the synthesis of antibodies that might cross
link the receptor and induce an anaphylactic response [4].
Also, most anti-IgE antibodies are also anaphylactogenic
and can cross link receptor bound IgE leading to the on-
set of allergic responses. On the other hand, the observa-
tion by [5], who described an anti-human-IgE mono-
clonal antibody (mAb) that did not induce histamine re-
lease, indicated the presence of IgE epitopes which are
obscured while engaged to the receptor and thus constitute
*The first three authors contributed equally to this publication.
#Corresponding author.
C
opyright © 2013 SciRes. JIBTVA

S. S. SABBAN ET AL. 16
valid anti-IgE targets only when free in solution. Several
non-anaphylactogenic mAb have since been described
that block the binding of IgE to its receptor [6,7].
Passive immunotherapy with non-anaphylactic anti-
bodies has demonstrated that it is possible to treat type I
hypersensitivity responses with mouse mAbs of which
Omalizumab [6] is the best characterized. The human-
ized antibody has been approved by the Food and Drug
Administration (FDA) and its efficacy has been demon-
strated in numerous clinical trials since 2000. The high-
affinity anti-IgE mAb, mAb12, can dissociate IgE from
its receptor by competition for binding sites [7,8]. Pas-
sive immunotherapy with administered anti-IgE antibod-
ies has, however, shown poor effectiveness in obese pa-
tients and in patients with IgE levels above 700 IU ml−1.
Furthermore, treatment only reduces symptoms tempo-
rarily, ~14 days, with 7% of individuals undertaking
Omalizumab therapy reporting adverse reactions. In ad-
dition, logistics and annual cost exceeds USD 50,000 per
patient.
The demonstration that some IgE-antibodies do not
evoke anaphylactic responses suggested that it might be
possible to devise a vaccine based on sequences in IgE
that elicit the synthesis of non-anaphylactic antibodies.
Such peptide immunogens can be expected to provide
long term protection against all IgE mediated allergies,
irrespective of the nature of the allergen evoking the IgE
response [4]. The determination of the co-crystal struc-
ture of the human IgE/FcεRIα complex [9], which identi-
fied epitopes in IgE-Fc region that become masked fol-
lowing receptor engagement, identified a region that
could be exploited to develop IgE-derived peptide vac-
cines that inhibit receptor sensitization and affect the
dissociation of receptor bound IgE [10-17].
Humans (hu), dogs (d) and horses (ho) are known to
suffer the clinical symptoms of IgE-mediated type I hy-
persensitivity responses. But no effective therapeutic
intervention strategies are currently available. Based on
an in vivo canine model system, several publications
[13,16,18] described the generation of an apparently ef-
fective therapeutic anti-IgE antibody response based on
the immunization with a chimeric IgE construct where
the canine Cε3 domain is flanked by sequences of Cε2
and Cε4 from opossum (Opossum-Dog-Opossum = ODO
Protein fragment). Following vaccination with this con-
struct, the authors report a reduction in blood IgE levels
of about 65%. But no information is available on the
binding affinity of the resulting anti-sera for canine IgE.
Furthermore, no assessment was made of the overall
immune response to canine Cε3 determinants, several of
which are potentially available after receptor docking and
could give rise to anaphylactogenic antibodies with po-
tentially lethal consequences.
Our current study assessed the immune response
against IgE-derived peptide epitope based on sequences
involved in the complementary interaction between hu-
man IgE and human FcεRIα and we obtained monoclonal
and polyclonal antibodies recognizing the inter Cε2-3
linker region, the AB helix and the FG loop. We subse-
quently focused on immune responses to peptides en-
compassing the inter Cε2-3 linker region since this epi-
tope is highly conserved between primates, horses and
dogs and may therefore have potential applications as a
universal vaccine to combat allergic responses in all
these species. Since anti-peptide antibody responses
commonly give rise to antibodies of low affinity, we de-
cided to test the potential applicability of the “Original
Antigenic Sin” [19,20] hypothesis to enhance the secon-
dary immune response. Rats were primed with the disul-
phide linked Cε2-3 linker region sequence (Fcε2-3 dimer,
Figure 1) and subsequently challenged with the same
peptide or a human Cε2-dog Cε3-human Cε4 chimeric
IgE (HDH) antibody construct (Figure 2).
To underpin these investigations we also developed
cellular assay systems to assess and test the safety of
anti-IgE immune responses directed against human, ca-
nine and equine IgE. Rat Basophil Leukemia (RBL-
2H3.1) cells which were transfected with the gene en-
coding human [21], canine [22] or equine [23,24] FcεRIα
and expressed the functional receptor complex on their
surface, could be sensitized with species specific IgE for
the assessment of both antigen and antibody induced
β-hexosaminidase release.
2. Materials and Methods
2.1. Design of the Disulphide Linked Fcε2-3
Dimer and the FG Loop Dimer
Peptides corresponding to the Cε2-Cε3 linker region, and
FG loop of the heavy chain of human and canine IgE
were designed and employed as immunogens, aiming to
provide the targets against which a specific anti-IgE an-
tibody response would be raised. The amino acid sequence
of these mentioned peptides are shown on Table 1.
As the peptide comprising the FG loop had to simulate
the native form as faithfully as possible, cysteine resi-
dues were introduced at both ends in order to establish a
Table 1. Peptide immunogens. End cysteine residues were
introduced in the FG loop peptide and were connected by a
disulphide bridge, resulting in formation of loop peptide
structure. Conserved residues are shown in bold.
HumanMW = 1005 DSTKKCADSNPRGVS
IgE Cε2-Cε3
Linker Region
Peptide CanineMW = 1050 DEARKCSESDPRGVT
HumanMW = 1134 CTHPHLPRAC
IgE FG Loop
Peptide CanineMW = 1150 CTHPHLPKDC

S. S. SABBAN ET AL. 17
Figure 1. Structure of the Fcε2-3 dimer showing the sequence
residue 323 - 336 of the human IgE molecule (which in-
cludes the Cε2-3 323 - 326 linker region and the 332 - 336
PRGV sequence found in human, canine and equine IgEs
highlighted in red) with a disulphide bridge at the cys 328
position, the dimer was then linked to the adjuvant thy-
roglobulin.
disulphide bridge constricting the peptides’ shape into a
loop form. Furthermore, a sample of each peptide was
covalently linked to KLH (Synthesis and Sequencing
Facility, the University of Sheffield), while another sam-
ple was mixed with MPL®/Tyrosine adjuvant (Allergy
Therapeutics Ltd) before they were used in the respective
mouse immunization protocols.
2.2. Generation of a Disulphide Linked Fcε2-3
Dimer
The Cε2-3 linker region peptide (Fcε2-3 dimer) encom-
passing residue 323 - 326 of the human IgE molecule
(Table 1) was chemically synthesized prior to dimeriza-
tion on a Millipore 9050 Peptide Synthesizer using Fmoc
chemistry. The linear peptide was purified by High-Per-
formance Liquid Chromatography (HPLC) and subjected
to oxidation in 200 mM NH4HCO3 at 4˚C for 72 hours at
a concentration of 5 mg·ml−1. The NH4HCO3 was re-
moved by freeze-drying and remaining traces of the bi-
carbonate were removed by freeze drying from water.
Correct assembly and oxidation (at cys 328 residue) of
the peptide was established by mass spectrometry. The
oxidized peptide was coupled to either keyhole Limpet
Haemocyanin or thyroglobulin via NH3 groups using
glutaraldehyde and mixed with MPL®/Tyrosine adjuvant
(Allergy Therapeutics Ltd) to give the final Fcε2-3 dimer
(Figure 1).
2.3. Generation of the HDH Anti NIP-HSA
Chimeric IgE Antibody Gene
The primers in (Figure 2) were used to amplify the re-
spective canine and human IgE Fc heavy chain domains
from canine IgE Fc heavy chain genomic DNA cloned
into the pCRII-TOPO plasmid [22] and human IgE Fc
heavy chain genomic DNA cloned into the pSV-VNP
plasmid by [25]. The required domains (the human Cε1,
Cε2 and Cε4 and the canine Cε3) were amplified by PCR,
and sub cloned into the pUC18 plasmid for sequence
verification by DNA sequencing. The domains were con-
structed to make the final HDH Fc heavy chain IgE gene,
which was then sub cloned into the pSV-VNP plasmid, to
make pSV-VNPHDH, where the HDH IgE Fc heavy chain
gene was inserted downstream of a mouse λ chain with
NIP specificity (Figure 2).
2.4. Generating the HDH Anti NIP-HSA
Chimeric IgE
The pSV-VNPHDH plasmid was transfected into J558L
cells (mouse B myeloma cells derived from BALB/c
strain [26]) by electroporation at 250v 960 μF (Bio-Rad)
and cultured using established procedures [27]. The
cloned cells were selected by Surface Plasmon Reso-
nance (SPR) and expanded for IgE expression.
2.5. Generation of Mouse Monoclonal Antibodies
Immunizations were performed under animal license
PPL 50/01317. Balb/c mice were immunized on days 1,
14, and 35 with 50 µg peptide in the presence of adjuvant.
Complete Freud’s adjuvant was used for the primary
injection, which was administered subcutaneously, fol-
lowed by intraperitoneal boost with incomplete Freud’s
adjuvant. Mice showing optimal titers were boosted with
100 µg of peptide in the absence of adjuvant. Spleens
were removed three days later to harvest spleen B cells
for the generation of hybridomas using established pro-
cedures [28].
2.6. Production of Anti-IgE Peptide Antibodies
in Rabbits
Rabbit polyclonal anti-IgE antibodies were produced by
GeneScript Corporation (New Jersey, USA) employing a
polyclonal express protocol and the proprietary T-Max
adjuvant. Prior to vaccination, blood samples were re-
moved from each rabbit as negative reference control.
Subsequently, three rounds of conjugated peptide vacci-
nations were made via intradermal and subcutaneous
routes. Serum was harvested from the rabbits displaying
the highest titers by exsanguination and the Ig fraction
was purified by Protein A column chromatography.
2.7. Rat Immunization Protocol
Immunizations were performed under animal license
PPL/40/3371. Male rats (Rattus norvegicus) aged ~10
weeks old were primed by subcutaneously injection with
100 μl of 1 mg·ml−1 of Fcε2-3 dimer (Figure 1) mixed
with an equal volume of complete Freund’s adjuvant,
followed by a subsequent injection with the dimer mixed
with incomplete Freund’s adjuvant 14 days later. 10 days

S. S. SABBAN ET AL. 18
The The non-thyroglobulin conjugated Fcε2-3 dimer was
used as a ligand to measure anti-serum antibody titers at
dilutions (1:200 - 1:102400). NIP-HSA was used to cap-
ture (human, canine or equine) IgE and to test binding of
the anti-sera to native IgEs, identical anti-serum dilutions
were used.
after the second injection ~200 μl of blood was taken
from the rat’s tail vein. After each bleed a minimum of
~20 days recovery was allowed before the rats were
boosted.
Test rats were subsequently boosted with 100 μl of 1
mg· m l−1 of HDH chimeric IgE antibody construct (Fig-
ure 2) mixed with an equal volume of incomplete
Freund’s adjuvant, while control rats where boosted with
the Fcε2-3 dimer. 10 days passed before a bleed was col-
lected (Table 2).
2.9. Cell Lines Expressing Human, Canine and
Equine FcεRIα
Cell lines transfected with genes encoding either human
[21], canine [22] or equine FcεRIα [23,24] were em-
ployed to assess the consequences of cell sensitization
and challenge with immune serum raised against the IgE
derived inter Cε2-3 dimeric peptide and HDH chimeric
IgE.
After each bleed, blood was incubated at room tem-
perature for ~1 hour followed by storage at 4˚C for ~24
hours before centrifugation at high speed (10,000 g for 1
minute). The resultant serum fraction was assessed for
anti-IgE titers.
2.8. Assay Protocol (ELISA) 2.10. β-Hexosaminidase Release Assays
Mediator degranulation assessed as β-hexosaminidase
release [21] was assessed using RBL-2H3.1 cells ex-
pressing a functional human, canine and equine FcεRIα
chain as described previously [2,3,22-24].
The mouse and rat anti-sera were analyzed using the En-
zyme-Linked Immunosorbent Assay (ELISA), using
immobilised peptide targets or native Fc IgE in order to
assess specificity and strength of the immune response.
Figure 2. PCR primers used to amplify and assemble the different domains of the human and canine IgE to construct the
final HDH IgE heavy chain gene (structure shown) having the human Cε1, Cε2 and Cε4 and the canine Cε3. The gene was
then inserted downstream of a mouse λ chain variable region with NIP-HSA specificity which resulted in a full HDH IgE an-
tibody with NIP-HSA specificity.
Table 2. The rat immunization schedule showing the pre-immunization bleed, the first and second bleed timings. Due to the
results of this investigation the third bleed was not preformed.
Bleed 1 Bleed 2 Bleed 3
Pre Immunization Bleed Injected on Day
0 & 14 Bled on Day 24Injected on Day 42Bled on Day 52Injected on Day 92 Bled on Day 102
Rat 1 Immunized with the Fcε2-3 dimer Boosted 1 with the Fcε2-3 dimer Boosted 2 with the Fcε2-3 dimer
Rat 2 Immunized with the Fcε2-3 dimer Boosted 1 with the Fcε2-3 dimer Boosted 2 with the Fcε2-3 dimer
Rat 3 Immunized with the Fcε2-3 dimer Boosted 1 with HDH IgE Boosted 2 with HDH IgE
Rat 4
No
immunization
Immunized with the Fcε2-3 dimer Boosted 1 with HDH IgE Boosted 2 with HDH IgE

S. S. SABBAN ET AL. 19
3. Results and Discussion tor s observencentratioti se-
rum (4.5 mg·ml−1, e 1:10 - 10,000).
dists
The protocol outlined in Sections 2.3 and 2.4 lead to the
ed HDH
anti ic IgE. After purification the chi-
de-
ction
2.8 ssessment of the immune response to the
ed recog-
nitio IgE, bind-
ing IgE was assessed. The outcome,
3.1. Binding of Anti-IgE Anti-Sera Directed
Against Human IgE Derived Peptides
The anti-peptide antibody responses were assessed by
SPR using the BIAcore 2000 system (General Electric
and results are summarized in Table 3).
Antibodies generated against the Fcε2-3 dimer (human
Cε2-3 linker region) recognized both the human and ca-
nine IgE-Fc with an affinity in the µM range.
When RBL-2H3.1 cells, transfected with human or
canine FcεRIα, were sensitized with cognate IgE and
challenged with Fcε2-3 dimer antiserum up to a concen-
tration of 50 µg·ml−1 no evidence was obtained for re-
ceptor cross linking. In contrast, IgE-mediated cell de-
granulation was observed in response to challenge with
the commercial anti-human IgE reference control, under
identical conditions, which supported β-hexosaminidase
release peaking at ~50% [27].
These observations clearly indicated that the immune
response to the Fcε2-3 dimer recognized human, canine
and equine IgEs and gives rise to non-anaphylactogenic
antibodies. Since the affinity of anti-peptide antibodies is
in the micromolar range, we assessed the potential of the
Fcε2-3 dimer antibodies/sera to prevent receptor sensitiza-
tion with human or canine IgE by investigating inhibition
of β-hexoseaminidase release following incubation of
human or canine IgE (1 µg·ml−1) with serial dilutions of
pre and post vaccination mAb or anti-serum for 1 hour
prior to sensitization of the respective transfected cell
lines, followed by a challenge with NIP-HSA antigen
(100 µg·ml−1) (data not shown). No inhibition of media-
Table 3. Binding of mAbs and rabbit antisera raised in re-
sponse to immunization of mice and rabbits with IgE-de-
rived peptides. Procedures leading to the generation of
mouse monoclonal and rabbit polyclonal anti-IgE derived
peptide antibodies are described in section 2.5 and 2.6. Ca-
nine and human anti NIP-HSA IgE were bound indirectly
to a CM51 chip via immobilized NIP-HSA and followed by
subsequent injections of each antibody, or serum, by pass-
ing over the chip.
Antibody Target KA (M)
Mouse IgG3 Human FG Loop 1.3 × 109
Mouse IgG3 Human AB Helix 2.4 × 105
Mouse IgG3 Human Cε2-3 Linker
Region 4.7 × 104
Mouse IgG2A Canine FG Loop 9.01 × 104
Mouse IgG1 Canine Cε2-3 Linker
Region 2.88 × 106
Rabbit Antiserum Human Cε2-3 Linker
Region 4.2 × 107 (Human IgE)
release wad at any con of an
serum dilution rang
inct binding siteSince several are known to con-
tribute to the docking of IgE to FcεRI, it is not surprising
that antibodies directed against a single peptide determi-
nant are unlikely to induce an antibody response of suffi-
ciently high affinity/avidity capable of inhibiting IgE/
receptor interaction or effect displacement of receptor
bound IgE. This is an important consideration in view of
the fact that most IgE is found complexed to cognate
receptors. We therefore decided to re-assess the observa-
tion by others [13,16,18], the results of which suggested
that immunization of dogs with a chimera encompassing
the complete canine Cε3 surrounded by opossum Cε2
and Cε4 domains (ODO) generated an immune response
capable of inhibiting allergic responses in dogs. We de-
signed a human Cε2-canine Cε3-human Cε4 chimeric
IgE antibody (HDH) to test this concept, although we
were aware that a complete Cε3 domain in context of
surrounding domains could generate an immune response
against Cε3 determinants which are not obscured when
the molecule is in contact with its receptor and may
therefore give rise to generation of anaphylactogenic anti-
bodies.
3.2. Generation of HDH Anti NIP-HSA
Chimeric IgE
development of a J558L cell line that express
NIP-HSA chimer
meric IgE was analyzed on a 12% SDS-PAGE (Figure 3)
in the presence and absence of the reducing agent β-
mercaptoethanol.
3.3. Binding of Rat Immune Serum to Fcε2-3
Dimer
The anti-Fcε2-3 dimer serum was raised in rats as
scribed in section 2.7 and tested by ELISA as in se
for the a
Fcε2-3 dimer. The results showed that sera from all four
rats recognized the Fcε2-3 dimer (Figure 4). Bleed 2 an-
tibody titers were higher than bleed 1. Variations in an-
tibody titer between rats were observed as expected.
3.4. Assessment of Rat Immune Serum
Reactivity to HDH Anti NIP-HSA
Chimeric IgE
Since the vaccine strategy employed the target
n of the Cε2-3 linker region of the native
to native canine
shown in Figure 5, indicated recognition of native canine
IgE with sera from rats 3 and 4 responding with higher
titer than rats 1 and 2, which were only boosted with the
Rabbit Antiserum Human Cε2-3 Linker
Region 7.1 × 106 (Canine IgE)

S. S. SABBAN ET AL. 20
Figure 3. SDS-PAGE of purified HDH anti NIP-HSA chi-
meric IgE in the absence (lane 2) and presence (lane 3) of
β-mercaptoethanol reducing agent. Lane 2 result shows the
predicted size of the full chimeric IgE (~192 kDa), whil
ere
lso assessed. The positive control was a rabbit anti hu-
at were boosted with the Fcε
di
ies in the rat sera, isolated from rats primed with
E,
-
gin iide may form the basis of a
20] where an in vivo im-
m
wn in Figure 6,
sh
it, thus aggregating the receptors on the cell
su
of human, canine and
eq
e
lane 3 shows the predicted sizes of the heavy (~70 kDa) and
lights chains (~25 kDa), the result also shows in lane 2 al-
most no bovine albumin impurity (~70 kDa) with minimal
protein degradation (bands at ~35 kDa and ~130 kDa).
Fcε2-3 dimer. Since the Fcε2-3 dimer comprises the PRGV
sequence, which is found in human, canine and equine
IgEs, responses to native human and equine IgE w
a
man IgE Cε3 anti-serum while the negative controls were
pre immunization sera.
Our results indicate that the immunization strategy in-
duced higher antibody titers for binding to native IgE
when rats were boosted with the chimeric HDH IgE
compared to animals th2-3
mer.
3.5. Assessment of Potential Anaphylactogenic
Immune Response
Antibod
the Fcε2-3 dimer and boosted with the chimeric HDH Ig
recognized native IgE of human, canine and equine ori
ndicating that this pept
common anti-allergy vaccine.
We further wished to establish whether the immune
response adheres to the principal of the “original anti-
genic sin”, a phenomenon commonly observed in re-
sponse to viral infections [19,
unization showed there is usually only one antibody
raised against one dominant epitope with traces of one or
two more. Upon re-infection, there is a tendency to make
antibodies against the first dominant epitope(s) encoun-
tered during the original exposure in spite of the presence
of new epitopes during subsequent encounters. Should
the second boost with the HDH chimera followed this
principle, a high titer of antibodies against the Cε2-3
dimer should result, and additional Cε3 epitopes, which
in principal, could be expected to stimulate the synthesis
of anaphylactogenic antibodies, and would not, in fact,
act as strong immunogens. The confirmation of such a
response could then form the basis for the development
of a safe anti-IgE vaccination schedule.
The sera, from bleed 2, of rats 3 and 4 were therefore
assessed for their capacity to induce β-hexosaminidase
release from cells sensitized with IgE as described in
Sections 2.9 and 2.10. The outcome, sho
owed that antibodies in the sera of the rats immunized
and boosted with the Fcε2-3 dimer did not induce media-
tor release. In contrast, sera from rats boosted with the
HDH chimera did cross link receptor bound IgE and
therefore activated downstream signaling from the FcεRI
receptor, resulting in mediator degranulation. Results
showed that this particular immunization protocol might
ultimately lead to the development of anaphylactogenic
antibodies.
The outcome of this experiment clearly shows that the
immunization protocol has resulted in the development
of antibodies that recognized receptor bound IgE and
cross linked
rface initiating downstream signaling resulting in de-
granulation and mediator release.
Our observations showed that immunization with a
peptide encompassing the Cε2-3 linker region known to
be involved in IgE/receptor [9] interaction gave rise to
antibodies capable of recognition
uine IgE. This suggests that further development of
this strategy could provide the basis for the development
of a pan-anti-allergic vaccine, provided that a safe and
effective high-affinity antibody response can be induced.
If successful, such an active immunisation strategy
would circumvent the problems associated with passive
antibody transfer, which are well reviewed in the litera-
ture. Even when the framework regions have been hu-
manised, the resulting antibodies, when used for passive
immunisation, may evoke immune responses to idiotypic
determinants. The advantage of the approach outlined in
this work is that it avoids this problem because it acti-
vates directly the species own immune system. The
maximal affinity observed in our study shows that the
immune response to the peptide is in the µM range, and
these antibodies, as we have shown, are incapable of in-
hibiting receptor sensitization, since the affinity of the
IgE for FcεRIα is in the nM range, reflecting the in-
volvement of multiple binding sites in the interaction and
the need to further improve the design of peptide targets
with respect to their similarity with their counterparts in
the native antibody. With respect to immunization with

S. S. SABBAN ET AL. 21
(a)
(b)

S. S. SABBAN ET AL. 22
(c)
(d)

S. S. SABBAN ET AL. 23
(e)
Figure 4. ELISA tests of both the first and second boost from each rat. All sera from immunized rats showed presence of an-
tibodies that bound to the Fcε2-3 dimer as predicted. Rats 1 and 2 were immunized with the Fcε2-3 dimer and this was used in
the subsequent boosts, while rats 3 and 4 were immunized, originally, with the same peptide but boosted with the HDH anti
NIP-HSA chimeric IgE. The pre immunization serum showed no binding to the Fcε2-3 dimer.
(a)

S. S. SABBAN ET AL. 24
(b)
(c)
Figure 5. ELISA results of second boosts from each rat. Sera were tested for binding to native human, canine and equine IgE.
Rats 1 and 2, which were boosted with the Fcε2-3 dimer, showed lower antibody titers than rats 3 and 4, which ere boosted
with the HDH anti NIP-HSA chimeric IgE. Rabbit anti human IgE Cε3 anti-serum was employed as a positive control and
pre immunization sera were used as negative control.
w

S. S. SABBAN ET AL. 25
Figure 6. β-hexosaminidase release from RBL-2H3.1 cells expressing canine FcεRIα after sensitization with ca- nine IgE and
challenged with antisera obtained after the second boosts of rats 1, 2, 3 and 4. The result shows that the immunization proto-
col has resulted in antibodies that recognized and cross linked receptor bound IgE, thus aggregating the receptors on the cell
surface resulting in degranulation and mediator release.
the HDH IgE chimera encompassing the original immu-
nogen, although it was shown to induce a strong immune
response to IgE in all three species, it is evident that the
generation of anaphylactogenic antibodies prohibits this
methodology from providing the basis of a potential
therapeutic strategy. It is interesting to note, however,
that in spite of the anaphylactic antibody response elic-
ited by the immunization schedule described in this study,
the immunized rats suffered no obvious side effects.
Similarly, there was no indication that the dogs immu-
nized with the chimeric ODO IgE described by [13,16,18]
suffering from adverse effects, although it is highly
probable that the immunization schedule described by
these investigators would have induced the formation of
potentially anaphylactic anti-IgE antibodies. Authors of
these publications fail to indicate whether this possibility
was investigated or even considered [13,16,18].
In order to improve the affinity/avidity of the immune
response, a cocktail of peptides representing sequences
of all the interactive sites between IgE and FcεRIα could
be administered together with novel adjuvants capable of
stimulating the synthesis of antibodies inhibiting and
reversing receptor sensitization.
The reagents described in the current study form the
basis for the initial in-situ safety testing of the immune
response to such immunogens, which in time, may have
an application in the prevention and treatment of all
IgE-mediated allergies by active immunization, irrespec-
tive of the nature of the allergen.
4. Acknowledgements
The authors wish to thank Allergy Therapeutics Ltd for
financial support and research materials and. L. J. Par-
tridge for her support with reagents and laboratory facili-
ties.
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