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Recent Advances in Immunotherapy for Allergic

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24

Recent Patents on Inflammation & Allergy Drug Discovery 2014, 8, 24-35

Recent Advances in Immunotherapy for Allergic Diseases

David El-Qutob*, Gemma Mencia and Enrique Fernandez-Caldas
Unit of Allergy, Hospital La Plana, Carretera Vila-Real-Burriana Km. 0.5 Vila-Real (Castelln) 12540, Spain
Received: June 8, 2013; Accepted: September 12, 2013; Revised: October 31, 2013

Abstract: Allergic diseases are a major health problem worldwide. The therapeutic approaches to treat allergic rhinitis
(AR) and allergic asthma (AA) fall in three major categories. The first step is allergen avoidance, or reduction of exposure
to the offending allergen(s). The second and most widely used therapeutic practice is the prescription of relevant medication to reduce symptoms. The third therapeutic element is specific allergy vaccination, also known as allergen specific
immunotherapy. Allergen-specific immunotherapy (SIT) is the only etiologic treatment of allergic disorders that can alter
the natural course of the disease. In this review, recent advances in immunotherapy and relevant patents are presented.
General vaccine modifications could be applied for any type of allergen. New specific modifications in allergic vaccines
have been developed for a variety of allergies such as house dust mites, horse, cat, parvalbumin and from birch, ragweed
and parietaria pollen.

Keywords: Allergoid, allergy, asthma, house dust mite, immunotherapy, parvalbumin, rhinitis.
INTRODUCTION
Rhinitis and asthma pose an important health problem
with a wide range of etiologies. Allergic rhinitis (AR) is an
IgE-mediated allergic disease that is a major health problem
worldwide [1]. AR is defined as a clinical symptomatic nasal
inflammatory reaction induced by specific IgE-mediated
mast cell degranulation after exposure of the nasal airways to
the offending allergens [2]. Furthermore, its prevalence has
been increasing in the last decades. Although AR in general
may not be a life-threatening disease, it usually progresses
into asthma, which annually causes a significant number of
deaths [3]. Furthermore, people suffering from allergic rhinitis show a lower quality of life than healthy individuals [4].
Asthma is a chronic inflammatory disorder of the airways
with participation of various types of cells [5]. It leads to
recurrent episodes of wheezing, breathlessness, chest tightness and cough, usually accompanied by variable airflow
obstruction, usually reversible with medication, as well as
spontaneously, and bronchial hyper responsiveness against
different stimuli.
There is an increasing interest in the presence and
movement of bioparticulate matter in the earth's atmosphere
and their impact on human health. The bioparticulates implicated to cause allergic symptoms are pollen grains, fungal
spores, insects, house dust mites, animal dander, chemicals,
foods, etc. [6]. Grass pollens and house dust mites are responsible for approximately 50% of all respiratory allergies
[7]. Of global importance are also animal dander (dog and
*Address correspondence to this author at the Unit of Allergy, Hospital La
Plana, Carretera Vila-Real-Burriana Km. 0.5 Vila-Real (Castelln) 12540,
Spain; Tel: 0034 964 3576 00; ext 54311; Fax: 00 34 964 357601; E-mail:
elqutob@comv.es; Tel: 964753600 ext 54310; Fax: 00 34 964 35 76 01;
E-mail: elqutob@comv.es

2212-2710/14 $100.00+.00

cat), other pollens (ragweed, mugwort, olive, parietaria,

birch) and mold (Alternaria, Aspergillus).
The therapeutic approaches of AR and allergic asthma
fall into three major categories. The first step is allergen
avoidance or reduction of exposure. Whereas specific allergen avoidance is easy to achieve in some cases of food allergies, it may be difficult or expensive for house dust mite
allergens, or it may be impossible, for pollen allergens. The
second and most widely used therapeutic option is the prescription of relevant medications in order to treat or minimize the symptoms, such as anti-histamines and corticosteroids. Symptomatic drugs are safe and effective; however,
they do not alter the natural evolution of the disease, neither
do they control the disease progression. The third therapeutic
step is specific allergy vaccination. Allergen-specific immunotherapy is the only etiologic treatment of allergic disorders
that can alter the natural course of the disease [8, 9]. It interferes with basic immunological mechanisms and induces
tolerance resulting in persistent reduction or even disappearance of the allergic symptoms. Thus, the protective effect of
specific allergy vaccination extends beyond the treatment
period in contrast to symptomatic drug treatment [10]. Furthermore, specific allergy vaccination has preventive effects
reducing the risk of hay fever developing into asthma, and
reducing the risk of development of new sensitivities [11,
12].
SIT involves administering gradually increasing
quantities of an allergen vaccine to an allergic subject, and
reaching a dose which is effective, improving the symptoms
associated with subsequent exposure to the causative
allergen. The usual route of administration is the
subcutaneous way (SCIT). In recent years, other ways have
been useful, such as sublingual or intralymphatic. Allergen
immunotherapy is indicated for patients diagnosed of allergic
2014 Bentham Science Publishers

New Allergic Vaccines

Recent Patents on Inflammation & Allergy Drug Discovery 2014, Vol. 8, No. 1 25

rhinitis and bronchial asthma and in allergic patients to

Hymenoptera venoms, who have specific antibodies (sIgE)
against clinically relevant allergens. Recently, there are
several studies focusing on food immunotherapy [13-16].

against allergens in many ways represents a key step in the

development of a healthy immune response against allergens
[26]. Several cellular and molecular mechanisms have been
demonstrated [27, 28]:

SCIT protocols, in general, involve two phases, the up

dosing and the maintenance phase. SCIT includes weekly
injections 8-16 weeks during an up dosing phase, followed
by monthly maintenance injections (empirically this has been
extended in some centres to 6-8 weeks) for a period of 3-5
years. Cluster immunotherapy up dosing schedules may
involve repeated injections at each clinic visit. Rush protocols which may involve repeated up dosing injections in order to achieve maintenance doses within several hours are
applicable to venom sensitive patients (native extract), although they are unsuitable for patients with inhalant allergies
in view of the marked increased occurrence of side effects.
In general, manufacturers' recommendations should be carefully followed, although tailored to individual patients'
circumstances [17]. In the up dosing phase, increasing doses
are applied, usually over a 16-week period, starting with
minimal concentrations of the allergen. When the
recommended maintenance dose is reached, this dose is
applied during the maintenance phase, typically involving
injections every 4 weeks. Following each injection, the
patient must remain under medical attendance for 30 minutes
due to the risk of anaphylactic side reactions, which in
principle although extremely rare could be life-threatening.
In addition, the clinic center should be equipped to support
emergency treatment [18].

Increase in peripheral blood of

suppressive capacities of:

For seasonal allergens, such as pollens, up-dosing is usually started and completed well in advance of the specific
season to avoid initiating treatment of patients with on-going
symptoms and hence to minimize the risk of side effects.SIT,
despite of its virtues, is not met with widespread use, primarily for two reasons. One reason is the inconvenience associated with the traditional vaccination program that comprises
repeated vaccinations. The other reason is that SIT may induce severe side-effects in allergic patients.
T cells play an essential role in the allergic response and,
therefore, the action by immunotherapy on these cells
represents a key mechanism in achieving an effective
treatment.The changes caused by immunotherapy are mainly
derived from an immune deviation from a Th2 phenotype
towards a Th1 phenotype, inhibition of antigen presentation
to specific T cells, and suppression of the activity of Th2
cells by regulatory T cells(Treg) [19, 20]. The induction of
IgG antibodies specific for the sensitizing allergens may inhibit IgE binding to antigens [21]. Specific Immunotherapy
induces an increase in certain IgG-antibodies [22]. These
IgG antibodies have been proposed as "blocking antibodies"
which compete with the binding of IgE to mast cells,
eosinophils, and other cells expressing IgE receptors. The
role of blocking antibodies is controversial. At present, the
production of IgG is considered an epiphenomenon, since the
increase of these antibodies occurs both in patients
responding to specific immunotherapy (SIT) as also in nonresponders [23]. However, some authors have shown that
IgG-antibodies could inhibit the formation of IgE-allergen
complexes [24, 25]. Several clinical trials of allergen-SIT
have demonstrated that the induction of a state of tolerance

allergen-specific

Inducible subsets of CD4+ CD25+ forkhead box

(FOX)P3+ T-regulatory

IL-10-secreting type 1 T-regulatory cells

Suppression of eosinophils, mast cells and basophils.

Antibody isotype change from IgE to IgG4.
For allergen immunotherapy, products may be either unmodified vaccines or vaccines modified chemically and/or
by absorption onto different carriers or adjuvants. There are
used aqueous (native extract), depot (absorbed) and modified
extracts (allergoids). Aqueous allergen vaccines can be used
for both venom and aero allergen hyposensitization. In the
depot extracts, the allergen is adsorbed to substances delaying its liberation such as aluminum hydroxide, calcium
phosphate or tyrosine. Allergoids consist of extracts of allergen polymerized to form larger aggregates with reduced allergenicity but with preserved immunogenicity. Formaldehyde and glutaraldehyde, separated or together, are frequently used to cross-link allergenic proteins to produce allergoids and have the major advantage of being less likely to
cause systemic reactions during immunotherapy than aqueous extracts. The allergoids enable to achieve the
maintenance dose more quickly and are safer [29].
Different approaches for specific allergy vaccination have
been performed to improve the safety and efficacy by adding
adjuvants, like Monophosphoryl lipid A (MPL), DNA sequences or bacteriophage combined with cytosine phosphodiester guanine (CpG) oligodeoxynucleotides (ODN), or by
modifying the allergen itself, or using recombinant allergens.
In these cases, T-cell epitopes should ideally be preserved so
that the resulting hypoallergen will still be able to modify the
allergen-specific immune response. Others have addressed the
route of administration such as sublingual (SLIT) [30] or intralymphatic (ILIT) [31] and the protocol regimen used.
Today, allergenic proteins can be identified and produced
in large quantities by recombinant DNA technology. Natural
allergen extracts can contain varying amounts of individual
allergens including allergens to which the patient may not be
sensitized. In the past years, recombinant allergen immunotherapy has offered a possibility to use well defined molecules with consistent pharmaceutical quality and defined in
mass units. Two studies are reviewed in an article published
in 2010 [32]. The first study applied a mixture of five
Phleum pratense major allergens in a maximum dose of 40
mcg protein. The clinical efficacy showed a significant reduction by 40% reduction in disease severity. The second
study compared a commercial birch extract with both recombinant Bet v 1 and purified Bet v 1 in dosages of 15 mcg
of allergen. The clinical result was 60% efficacy. The advantages of using recombinant allergens for immunotherapy
need more studies before the overall value in terms of efficacy and safety can be assessed.

26 Recent Patents on Inflammation & Allergy Drug Discovery 2014, Vol. 8, No. 1

In the present paper, recent attempts to improve treatment

with allergic vaccines will be reviewed, particularly modifications of extracts, use of adjuvants and recombinant allergens, and alternatives routes to SIT.
PATENTS OF SPECIFIC-ALLERGEN VACCINES
1. House Dust Mite Allergy Immunotherapy Modifications
Hypoallergenic Polypeptides for the Treatment of House
Dust Mite Allergy
House dust mites (HDM) are one of the most important
risk factors associated with the development of allergic diseases such as rhinitis, asthma and conjunctivitis [33]; more
than 50% of all allergic patients worldwide suffer from
HDM-allergy. Mites belong to the arthropod group and have
a size of less than 0.3 mm. The species that are most often
implicated in allergic cases are those belonging to the genus
Dermatophagoides. Their optimal growth conditions include
temperature of about 25C and relative humidity above 65%.
To date, 24 allergens from the most common house mites D.
pteronyssinus and D. farinae have been described.
Allergens of the group 1 (Der p 1) are proteins with cysteine protease activity. Natural Der p 2 is a mixture of isoforms that differ notably at amino acid positions 40, 47, 127
at 114 [34]. Der p 1 and Der p 2 react with 80-100% of
HDM allergic patients [35].
Several hypoallergenic derivatives of group 2 mite allergens have already been developed and have been shown to
be suitable for immunotherapy [36, 37]. However, only a
few hypoallergenic derivatives of group 1 mite allergens
exist, which are not well characterized [38]. Asturias et al.
[39] in 2009 studied hypoallergenic derivatives referred as
QM1 and QM2. QM1 was obtained by the fusion of both
proteins (Der p 1 and Der p 2) and the elimination of one
Disulphide Bridge. QM2 was obtained by the insertion of the
protein Der p 1 between the residues 73 and 74 of Der p 2.
Allergenicity of QM2 was practically zero, and QM1 exhibited 50 times lower allergenicity than that obtained with the
natural allergen. Both QM1 and QM2 maintained immunoproliferating capacity, which was even greater in the
fused proteins compared with wildtype proteins. Bussieres
[40] in 2010 described studies on recombinant fusion proteins assembling Der p 1 and Der p 2 but with a modest reduction of approximately 10-fold of their allergenic activity.
Fusion proteins encompassing Der p 2 with either mature or
proDer p 1 were expressed in Escherichia coli or Pichia pastoris. Chen et al. published the reduction of allergenicity of
Der p 2 only [36], excluding the use of such derivatives to
treat Der p 1-allergic patients. Authors synthesized two recombinant fragments of Der p 2 comprising amino acids 153 and amino acids 54-129 and a hybrid molecule (amino
acids 54-129 + 1-53), combining the two fragments in inverse order, by genetic engineering.
The authors of this invention created a hypoallergenic
combination vaccine for immunotherapy in HDM allergic
patients [41]. The two constructed mosaic proteins consisting
of fragments derived from Der p 1 and Der p 2. One construct contained the original amino acids of the two wildtype

El-Qutob et al.

allergens (Der p 2/1C) whereas in the other construct cysteine residues were replaced with serine residues (Der p
2/1S). These two mosaic proteins are characterized by an
almost complete lack of IgE reactivity and allergenic activity. Dot-blotted nDer p 1, rDer p 2, the two Dp 2/1 mosaic
proteins and BSA were tested for IgE reactivity with sera
from 21 HDM-allergic patients, serum from a non-allergic
individual and buffer without serum. Bound IgE were detected with labeled anti-human IgE antibodies and visualized
by autoradiography. Basophils from 8 mite allergic patients
were stimulated with various concentrations of nDer p 1,
rDer p 2, Der p 2/1C and Der p2/1 S. Expression of CD203c
was determined by FACS analysis and is displayed as stimulation index.
This reduction of allergenicity is a very interesting modification of allergy vaccines but it is necessary to carry out
studies of safety and efficacy. In this sense, the modification
suggested by Asturias et al. could be helpful. However, we
should bear in mind, that patients could be allergic to more
than a single HDM protein. In those cases, these very specific vaccines will not work so efficiently because these vaccines will lack some allergens to which patients are sensitized.
Recombinant Der p 2 Expressed in Pichia pastoris as a
Natural-like Allergen for Immunotherapy and Diagnostic Purposes
The present invention provides a method for producing a
recombinant D. pteronyssinus protein (rDer p 2), comprising
the steps of cultivating a Pichia pastoris yeast strain previously transformed with a rDer p 2 coding sequence and isolating the rDer p 2 protein [42]. Isolation from the culture
supernatant or from cellular extracts consists of chromatography, followed by dialysis and concentration. Sublingual
immunotherapy (SLIT) with rDer p 2 decreases airway hyperresponsiveness (AHR) reduces lung eosinophilia and
lowers nDer p 2-specific Th2 cells responses in nDer p 2sensitized animals.
This invention is very interesting because previous attempts to produce a recombinant Der p 2 protein similar to
its natural counterpart have failed, yielding at best a molecule with partial folding, This invention was successful in
achieving this for the first time.
2. Contiguous Overlapping Peptides (COPs) for Treatment of Birch Pollen Allergy
Approximately 25% of all allergic patients respond to
tree pollen and among those, 90% show reactivity with birch
pollen extract on skin prick test. Most patients show hypersensitivity to Bet v 1, the major birch pollen allergen. Bet v 1
is part of a protein family playing an important role in plant
defense (Pathogenesis-related proteins or PR-proteins) and
thus Bet v 1 cross -reacting proteins were found in a number
of plants [43]. So, there is cross-reactivity between birch
pollen and some foods as hazelnut, peach, melon and apple
[44, 45]. The birch genome contains at least 7 pollenexpressed genes that encode distinct Bet v 1 isoforms with
varying IgE reactivity. The isoform Bet v 1.0101 has show
the highest IgE reactivity. In contrast, the isoforms Bet v
1.0401 and Bet v 1.1001 present very low IgE reactivity, and

New Allergic Vaccines

Recent Patents on Inflammation & Allergy Drug Discovery 2014, Vol. 8, No. 1 27

higher stimulatory capacity for T cells than Bet v 1.0101.

Therefore, these isoforms have been considered promising
candidate molecules for SIT [46]. A clinical trial with recombinant Bet v 1 has shown efficacy equivalent to whole
birch pollen extract [47]. Studies show that an efficient immunotherapy product should preferably contain the complete
sequence of the allergen rather than select T-cell epitopes.
Therefore, Bauer et al. [48] evaluated in mice a DNA-based
vaccine encoding Bet v 1 stably linked to ubiquitin. This
vaccine specifically suppressed IL-5 induction and airway
eosinophilia with significant downregulation of IgE and with
a slight delay regarding establishment of maximum suppression compared with the positive control.
Two contiguous fragments or Bet v 1 or trimeric forms of
Bet v 1 were tested in a phase I study in human and showed
a trend towards improvement in symptom medication scores
[49] but with increased number of adverse events [50]. This
investigational group investigated whether IgG antibodies
induced following vaccination with genetically modified
hypoallergenic Bet v 1 derivatives were able to inhibit IgEfacilitated binding of allergen-IgE complexes to B cells with
positive results [51].
The present invention consists of repeatedly administering specific COPs to humans suffering from birch pollen
allergy [52]. Such peptides can be obtained by any of a variety methods including by chemical synthesis or by recombinant means. Authors conducted a single randomized placebo-controlled phase I/IIA clinical trial including volunteers
with birch pollen AR and asthma to evaluate AllerT. AllerT
is a product composed of a set of three COPs derived from
the major birch pollen allergen Bet v 1. Overall AllerT was
safe (Basotest negative, local adverse events were mild and
did not differ from placebo), increased specific- IgG4 and
IL-10 and improved MiniRQLQ and asthma symptom score
compared to placebo.
It is desirable to find vaccines that induce a safe immune
response and this product is a promise of future. Treatment
with AllerT must wait until clinical trials are finished and
results are published.
3. Hybrid Proteins from Parietaria judaica Major Allergen and Uses Thereof
Parietaria pollen is one of the most important causes of
allergy in the Mediterranean area. The two main allergens of
Parietaria judaica pollen are Par j 1 and Par j 2. These are
proteins have a percentage of sequence identity around 45%
and is more prominent in the N-terminal region of the proteins, where the most important IgE epitope is located. Par j
1 and Par j 2 are non-specific Lipid Transfer Proteins
(nsLTPs). Par j 2 can be used as a marker for the diagnosis
of Parietaria allergy [53]. Other patents [54] have presented
hypoallergenic variants of major allergen Par j 2 with a decrease in binding capability to specific IgEs. Bonura et al.
[55] in 2007 tested the Par j2-par j 1 hybrid by comparing it
with its derivative structure. The hybrid has reduced IgE
reactivity maintaining its T epitopes unchanged because,
when injected into mice, it is able of inducing an IgG production capable of recognizing the single Par j1 and Par j2
allergens. Gonzalez-Rioja et al. developed a hybrid with
only 29-52 fragments containing the 4 cysteines involved in

the four disulphide bonds. This hybrid had lower IgE reactivity compared to a mix of the two singles allergens and the
ability to stimulate T lymphocytes remained unaltered compared to the mix of the single proteins, while it significantly
decreased in the case of other hybrid.
The present invention provides a hybrid protein consisting of mutated Par j 1 and Par j 2 sequences linked to each
other [56]. When incubated with the sera of twelve Parietaria
pollen allergic subjects by ELISA assay, the hybrid protein
exhibited a mean decrease in the reactivity towards IgEs of
41% compared to wildtype hybrid. Moreover, hybrid molecules induced an earlier IgG response to Parietaria extract
compared to the whole extract. Further studies are needed to
validate this type of immunotherapy.
4. Vaccine Peptide Combinations Against Cat Allergy
Approximately 10% of the general population in industrialized countries have allergy to cats (Felis domesticus).
The major allergen produced by cats is the glycoprotein Fel
d 1, which belongs to the uteroglobin protein family and
elicits a response in 90-95% of patients suffering from cat
allergy. This 39 kDa protein is formed from two 17 kDa
subunits, each consisting of two disulphide-linked peptides.
The major source of the Fel d 1 protein is the sebaceous
glands, although expression is also detected in salivary
glands and the anal glands [57]. Zhu and colleagues [58]
have developed a strategy where they target the inhibitory Fc
receptor FcRIIb on mast cells and basophils. In contrast to Fc
RI, which activates these cells, FcRIIb has the ability to inhibit activation of the cell response. Hulse et al. [59] linked
recombinant Fel d 1 to a single chain fragment of a humanized anti-CD64 monoclonal antibody targeting the resulting
fusion protein (termed H22-Fel d 1) to the high-affinity
receptor for IgG (FcRI) on APCs. Compared with Fel d 1
alone, H22-Fel d 1 induced increased numbers of IL-10+ and
IL-5+ CD4+ cells. Modification of the T-cell response was
only observed in cells from patients with allergy. Grnlund
et al. [60] developed a new adjuvant and allergen delivery
system for SIT and applied this approach on Fel d 1. It is
based on covalent linkage of the allergen to agarose particles
(CBP) with a size of 2
m in diameter. The treated mice exhibited less AHR, decreased levels of Fel d 1-specific IgE
and increased levels of IgG compared with placebo treated
mice. Nilsson et al. [61] published a study in mice with random mutations of rFel d 1 that were isolated, displaying
similar or lower IgE binding, reduced anaphylactic activity
as measured by their capacity to induce basophil degranulation and a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. These
peptides cause T cell proliferation with minimal histamine
release. An allergen-specific immunotherapy with recombinant fusion proteins derived from hepatitis B virus and 2
nonallergenic Fel d 1 peptides has also been evaluated. This
approach showed a lack IgE reactivity and IgE-mediated
allergenic activity and exhibit reduced T-cell reactivity [62].
Grundstrm et al. [63] treated mice sensitized to Fel d 1 with
SCIT of two doses of recombinant Fel d 1 coupled to 1, 25,
dihydroxyvitamin D3 (rFel d 1:VD3) and compared to
treatment with the same doses of rFel d 1 in a mouse model
for cat allergy. Treatment with both doses of rFel d 1:VD3
decreased airway hyperresponsiveness (AHR), cellular in-

28 Recent Patents on Inflammation & Allergy Drug Discovery 2014, Vol. 8, No. 1

flux and Th2 cytokines in BAL compared to untreated mice.

But only were differences in the total number of cells and
eosinophils in BAL when comparing rFel d 1:VD3 and rFel
d 1 alone.
Senti et al. [64] published in 2012 a randomized doubleblind controlled with placebo trial using intralymphatic immunotherapy (ILIT) with modified Fel d 1 in alum. ILIT
induced 74-fold increase in nasal tolerance after only 3 injections. Martinez-Gomez [65] published in 2009 that the use of
ILIT induced more than 10-fold higher IgG2 responses with
100-fold allergen-doses than SCIT. Worm et al. [66] identified immunodominant MHC-binding sequences of Fel d 1
that administered were safe and well tolerated as a single
dose of 3 nmol. Saarne et al. [67] treated mice with hypoallergen of rFel d 1 that previously shown to have retained Tcell reactivity and strongly reduced IgE-binding capacity
compared to unmodified rFel d 1. This immunotherapy was
well tolerated, produced increased serum levels of rFel d 1specific IgG1 and IgG2 compared to placebo treated mice
but high treatment doses were required to reduce AHR.
These authors constructed in 2005 three Fel d 1 hypoallergens with reduced IgE-binding capacities and retained T cell
reactivities.
The present invention is useful and effective in reducing
hypersensitivity to Fel d1 allergen in individuals that are
sensitized to this allergen [68]. The peptide combinations
presented bind to many different MHC Class II molecules,
produce the same or less histamine release than the whole
allergen and/or have a cytokine release profile equivalent to
the whole allergen, and are soluble. The individual to be
treated is from a population that has MHC allele frequencies
within the range of frequencies that are representative of the
Caucasian population. According to the authors, the current
invention also provides means of desensitizing individuals
that are allergic to multiple allergens and can create in the
individual a tolerogenic environment wherein inappropriate immune responses to other allergens can be downregulated in order to provide tolerance to other antigens. Authors
think that the present invention can also benefit patients with
autoimmune disorder and food allergy. The present peptide
combination was analysed in 94 subjects measuring Interferon-
(IFN-
), IL-13 and IL-10 productions. IFN-
responses were detected between 26-44% of subjects in response to individual peptides. IL-13 responses were detected
in between 33-68% of subjects in response to individual peptides. IL-10 responses were detected in between 46-75% of
subjects in response to individual peptides. A preferred mixture of 7 peptides was tested in a randomized, placebocontrolled, blind clinical trial. Two intradermal injections of
commercially available standard cat allergen were administered and subjects were assessed to ensure that they experience a Late-Phase Skin Response (LPSR) to whole cat allergen. The treatment Phase consisted of a period of 21 days for
each subject. During this period one group of subjects received a single intradermal injection of either the preferred
mixture or diluent placebo. Authors indicated that the mixture of peptides is effective at reducing the LPSR to whole
allergen in cat allergy individuals.
The percentage of sensitization to cat that authors has
presented is not correct. That percentage belongs to the

El-Qutob et al.

population of industrialized countries and not to the world's

population [69, 70].
The tolerogenic effect of the invention is a known effect in respiratory allergic patients receiving immunotherapy.
But there is not a possible effect over autoimmune disorders
or food allergy. Unfortunately, only Caucasian population
can be treated with this peptide combination. It is necessary
more studies to assess the efficacy and safety of this product.
5. Vaccine Comprising Amb a 1 Peptides for use in the
Treatment of Ragweed Allergy
Common Ragweed (Ambrosia artemisifolia) represents
the most prominent seasonal allergen in North America and
Mexico. Ragweed pollinization comprises from the end of
June to the end of September. Ragweed pollen contains several allergens, of which Amb a 1, a 38 kDa protein belonging
to the pectate-lyase protein family, is the most important.
More than 90% of ragweed-sensitized subjects react to Amb
a 1 in skin prick tests, and at least 90% of the allergenic activity in ragweed pollen can be attributed to this protein [71].
Four isoallergens of Amb a 1 with 70% to 80% amino acid
sequence identity have been identified [72]. T-cell epitopes
presented by HLA-DP or HLA-DQ molecules, as observed
in Amb a 1, might sensitize a large part of the population
[73]. The use of toll-like receptor (TLR) ligands in combination with allergens to modify the function of APC has been
proposed as a new strategy for biasing the immune response
toward Th1 in allergic disease [74]. Amb a 1-immunostimulatory phosphorothioate oligonucleotide conjugate (AIC)
has received a lot of attention in recent years [75-77] and has
lately undergone large multicenter phase 2/3 clinical trials
with encouraging short- and long-term clinical results in subjects with moderate-to-severe ragweed allergy. It is produced
by covalently linking the purified short ragweed pollen allergen, Amb a 1 conjugate, to a phosphorothioate oligodeoxyribonucleotide immunostimulatory sequence (ISS-ODN) containing a CpG motif. The ISS is a short synthetic DNA sequence that binds to TLR9 on plasmacytoid dendritic cells
(pDC) with subsequent activation of the innate immune system. Amb a 1-ISS conjugate has an enhanced Th1-biased
immunogenicity and reduced allergenicity. It may offer a
more effective and safer approach for allergen immunotherapy than currently available methods.
Authors present polypeptides selected to retain T cell
specificity whilst not being able to cause significant histamine release) [78]. These polypeptides induce a cytokine
response in a high proportion of ragweed allergic individuals
(Multiplex bead array assays) and are soluble. The peptides
provided by the present invention may be derived from
splice variants of the parent proteins encoded by mRNA
generated by alternative splicing of the primary transcripts
encoding the parent protein chains. The peptides may also be
derived from amino acid mutants, glycosylation variants and
other covalent derivatives of the parent proteins which retain
at least an MHC-binding property of the allergens.
6. Novel Therapies for Grass Pollen Allergic Patients
Grass pollen is a major cause of allergic disease that may
affect approximately 20% of the general population [79],
severely affecting the quality of life of these patients and

New Allergic Vaccines

Recent Patents on Inflammation & Allergy Drug Discovery 2014, Vol. 8, No. 1 29

causing huge costs to society [80]. Ohlin et al. [81] provide

hypoallergenic polypeptides comprising of a variant of the
amino acid sequence of the C-terminal sequence of the major
timothy group 1 pollen allergen Phl p 1 wherein one or more
amino acids of this sequence within an IgE-binding epitope
is mutated and wherein the polypeptide exhibits reduced
IgE-reactivity compared to a polypeptide consisting of the
unchanged amino acid sequence. The polypeptides are linked
to polymers which increase stability and half-life (e.g. polyethylene glysol- PEG) and this attachment not impair the
ability of the polypeptide to induce the formation of IgG
antibodies to Phl p 1. Authors measured cross-reactivity of
Phl p 1-binding human monoclonal IgE against protein extracts of 10 grass species with group 1 allergens with high
sequence identity to Phl p 1 determined by ImmunCAP.
Also, authors determined by ELISA the reduced reactivity of
serum IgE from Phl p 1 allergic donors against the potentially hypoallergenic variant.
This patent is interesting because provides pharmaceutical compositions of such polypeptides, as well as methods
for making and using these polypeptides, for treating pollen
grass allergies. Reduce the allergenicity keeping the immunogenicity is a fundamental premise in the use of immunotherapy in allergic diseases.
PATENTS
CINES

OF NON-SPECIFIC-ALLERGEN

VAC-

1. Use of an Adjuvanted Allergy Vaccine Formulation for

Parenteral Administration
In general, in seasonal allergens such as pollen, a preseasonal start of immunotherapy is recommended as explained above. The patient must start the treatment when he
or she is free of symptoms. An intra-seasonal start of the
immunotherapy would allow entering more patients into
causal treatment. The up-dosing phase is initiated after start
of the allergen season. Authors considered that season starts
the first of three consecutive days wherein the level of airborne allergen is above a threshold value which is 5% of the
average peak value of the previous 10 years, at one or more
measuring locations in the region of country. Authors have
investigated the safety of an intra-seasonal start of immunotherapy with two types of vaccines: an aluminum hydroxide
adjuvanted depot vaccine for long term treatment (ALKDepot SQ) and an aluminum hydroxide adjuvanted depot
vaccine for short term treatment (ALK7) [82]. A total of 134
patients intra-seasonally were treated with ALK-Depot SQ
and 12 patients with ALK7.
Only 6 systemic reactions (SR) with nausea in the same
patient with intra-seasonal up-dosing immunotherapy were
documented. The incidence of large local reactions (LLR) is
similar between pre-seasonal start and intra-seasonal updosing. LLR are more frequent in intra-seasonal up-dosing
but SR are less frequent. The study showed that an intraseasonal start of immunotherapy is safe for aluminum hydroxide based subcutaneous allergy vaccines.
Current state of the art recommends the immunotherapy
must start pre-seasonally but with more studies showing
good safety and efficacy it could change. Today, there are
vaccines administered pre-seasonally only few weeks before

with good efficacy and safety. It could be a first step to

change the moment for starting immunotherapy, preseasonally or intra-seasonally.
2. Hypallergenic Mosaic Antigens and Methods of Making Same
In this patent, authors provide a method of making a reassembled mosaic antigen including the steps of cleaving a
naturally-occurring allergen into non-overlapping peptide
fragments and recombining them in such a way that the mosaic antigen includes all, or substantially all, of the amino
acids of the original naturally-occurring allergen, though in a
different order [83]. These mosaic antigens have a reduced
or eliminated capacity to bind IgE while conserving at the
same time those features of the allergen that are required to
induce a T-cell mediated immune response. The reassembled
mosaic antigens of the present inventions are capable of inducing a strong allergen-specific IgG response while simultaneously inhibiting or suppressing IgE production. Authors
present an example with timothy grass pollen (Phl p 1 and
Phl p 2) and birch pollen (Bet v 1). The IgE binding capacity
of purified Phl p 2 mosaic (P2M) was compared with that of
Phl p2 wild-type (P2) by dot blot experiments using sera
from twelve timothy grass pollen allergic patients. The reduced allergenic activity of the rPhl p 2 mosaic was further
demonstrated by basophil histamine release and skin test
experiments.
3. An Allergen Dosage Form
SLIT consists of the periodic dosing of a solution of the
allergen at intervals spaced apart by at least one day. Authors
present a fast-dispersing non-compressed solid dosage allergen without undesirable level of side effects and without to
incorporate an adjuvant in order to enhance the immunestimulating properties of the sensitizing allergen [84]. A
study with forty-seven allergic patients to Phleum pratense
pollen was carried out. Patients received a daily sublingual
dosage for a period of 8 weeks. Four groups were treated
with 2500 SQ, 25000 SQ, 75000 SQ and placebo respectively. The SQ-Unit is determined in accordance with ALKAbell A/Ss "SQ biopotency"-standardisation method
where 100,000 SQ units equal the standard subcutaneous
maintenance dose. The doses were well tolerated in all three
active treatment groups as adverse events were predominantly mild in severity and limited to itching in the mouth
and throat. The results also showed that the formulations are
stable in respect of friability as allergen release was detected.
In the past years, SLIT is a good alternative to SCIT in
allergic patients. Modifications could be applied to improve
the safety and efficacy of SLIT and should be considered
actually. In this case, more studies are needed to assess the
results showed.
4. Allergoid Derived from Allergens
The therapeutic efficacy of SIT has been shown in a
number of clinical studies, it is not free from risks related to
the also severe undesired reactions [85, 86]. In recent years,
a great deal of attention has been focused on the development of vaccines that are more or less selective, yet aimed to
reduce the allergenic potential of the vaccines by preservat-

30 Recent Patents on Inflammation & Allergy Drug Discovery 2014, Vol. 8, No. 1

El-Qutob et al.

ing their immunogenic potential as much as possible: the

development of the so-called allergoids.

For the determination of cytokines and chemokines of the

innate immunity, commercial ELISAs have been used.

The authors of the present invention propose modified

allergens that have a reduced allergenicity (by EASTinhibition, immunogenic ability by ELISA, and molecular
dimensions by SDS-PAGE) compared to the corresponding
native allergenic material and characterized in that all or a
part of the primary amine groups of the lysine and arginine
residues of the allergenic molecules are functionalized as
shown in the structure, said allergens assuming, after modification respectively with carbamylation or thiocarbamylation
reaction, and reaction with a dialdehyde or diketone [87].
The functionalization by a carbamylation or thiocarbamylation reaction occurs by treatment with alkaline cyanate
(KCNO or NaCNO), or organic isocyanates or tiocyanates.

This patent opens a new path to improve a change in the

phenotype and also decrease the inflammatory mediators that
are released during allergic reaction.

This invention is an interesting modification of allergic

vaccines because it proposes a preparation for specific immunotherapy (SCIT) that provides with a higher tolerability
and minimizes the risk of possible undesired effects without
thereby decreasing the desensitization effect which is intrinsic of the conventional allergenic therapy.
5. Preparations of Conjugates Comprising Adenine Derivatives and Allergenic Proteins and their use for Specific Immunotherapy of Allergenic Diseases
At present several products for specific immunotherapy
are available on the market for the treatment of allergic diseases. However, SIT exhibits several limitations related to its
profile of efficacy and safety. SIT can determine an exaggerate release of mediators due to the stimulation of FceRI+
cells because of the administration of allergenic extracts to
the patients with the possible consequence of severe adverse
effects including anaphylactic shock.
The authors of the present invention created adenine derivative active ester and use thereof for the preparation of
stable conjugates between adenine derivative and allergenic
proteins for the modulation of Th2 response in allergic diseases [88]. The conjugation of a protein antigen together
with modified adenines are able to modify the allergenspecific response with re-direction towards a less pathogenic
phenotype and exhibiting lower ability to stimulate the release of mediators of the allergic inflammation. Redirected
response is determined by the synergistic activity of the antigen and the adenine derivative, possibly a cause of the covalent conjugation between the protein of interest and the
modified adenine. Adenine derivative has initially been functionalized with a linker by treatment with ethyl 4bromobutanoate in anhydrous dimethylformamide and the
presence of K2CO3 as a base. The product is hydrolyzed in
alkaline medium with KOH in a 3:1 methanol-water mixture.
After 48 h, the methanol is evaporated and pH adjusted by
acid. Finally, the active ester is prepared by dissolving the
acid in anhydrous dimethylformamide and adding dicyclohexylcarbodiimide and N-hydroxysuccinimide. After chromatography of the crude reaction mixture the active ester is
obtained. The conjugation with the allergen is carried out by
employing an excess of active ester compared to the moles
of lysine evaluated to be present on the amount of the protein
used.

6. Suppression of a Hypersensitivity Immune Response

with Unrelated Antigen Derived from Allergen Source
Material
Several researchers have investigated bystander suppression to an immune response by administering an antigen
unrelated to the antigen triggering the immune response [8991]. In contrast to conventional allergen specific immunotherapy where the individual is treated with the specific allergen triggering the hypersensitivity immune response, the
present invention relates to the treatment of a hypersensitivity immune response in an individual with an antigen unrelated to the allergen triggering a hypersensitivity immune
response in the individual. That is to say that the present invention also relates to bystander suppression of a hypersensitivity immune response. The inventors have now found that
co-exposure might be provided by selecting an unrelated
antigen that is naturally exposed to the target organ along
with the antigen triggering the hypersensitivity immune response in the individual) [92]. Two different allergens, OVA
(ovoalbumin) and Phl p extract (Phleum pratense) have been
used as exemplary unrelated antigens. As a first step, nave
mice receive SLIT with the unrelated antigen in order to induce tolerance. Subsequently, the effect of the SLIT treatment is evaluated by inducing a systemic immune response
by injecting the triggering antigen adsorbed to alum intraperitoneally. The immune response consists in spleenic Tcell proliferation and production of the Th2 cytokines upon
in vitro re-stimulation with the allergen.
7. Allergen Peptide Fragments and use Thereof for
Treatment of Dust Mite Allergies
The authors of the present invention provides a composition containing a plurality of contiguous overlapping peptide
(COPs) fragments which together comprise the entire amino
acid sequence of a chimeric dust mite allergen, wherein the
fragments are capable of inducing a T cell response in patients who are hypersensitive to the allergen [93]. The administration of this composition results in lower level of IgE
stimulation activity and/or IL-4 production than the amount
of IgE and/or IL-4 production stimulated by the whole protein allergen. IL-4, IL-10 and IFN
are titrated using commercially available ELISA kits. This method is useful in
treating a number of different allergies to various allergens
(HDM, plant pollens, grass pollens, tree pollens, weed pollens, insect venom, animal dander, fungal spores and food
allergens).
This invention demonstrates that allergen fragments can
induce tolerance with SIT.
8. Carbon Nanotube Compositions and Immunotherapy
Polymeric nanoparticles are colloidal carriers that vary in
size from 10 to 1000nm [94]. Biodegradable polymers are the
most used as great promise the field of drug-delivery systems.

New Allergic Vaccines

Recent Patents on Inflammation & Allergy Drug Discovery 2014, Vol. 8, No. 1 31

These types of nanoparticles provide controlled/sustained release properties, subcellular size, and biocompatibility with
tissues and cells [95], and they are well established carrier
systems with high potential for the delivery of bioactive macromolecules, including peptides, proteins, and nucleic acid
vaccines. Nanoparticles can be designed to provoke an immune response, by either direct immunostimulation of antigen
presenting cells or delivering antigens to specific cellular
compartments [96]. To achieve the desired therapeutic effect
with these biodegradable polymeric devices, it is important, to
select an encapsulating agent, since its nature significantly
influences the size and the release profile of the nanoparticles
[97]. These biodegradable polymers can be either natural (chitosan, alginate, carrageenan, albumin, gelatin, collagen,
among others) or synthetic poly-lactic acids, poly-lactide-coglycolic acids, poly-methyl methacrylate, poly-
-caprolactone,
poly-alkylcyanoacrylates, and copolymers [98].
Currently, nanoparticles-based allergen-delivery systems
have received much interest as potential adjuvants for allergen immunotherapy. It has been demonstrated that incorporation of allergens into a delivery system plays an important
role in the efficacy of allergy vaccines. In the last years, several nanoparticles-based delivery systems have been described, including biodegradable and nonbiodegradable polymeric carriers [99, 100].
In the patent US20130216581 [101], authors present carbon nanotube (CNT)-based compositions for activating immune responses. The CNT compositions function as artificial
antigen-presenting cells (aAPCs) or as modular vaccines.
The disclosed CNT aAPCs are efficient at activating T cells
and may be used to activate T cells ex vivo or in vivo for
adoptive or active immunotherapy. CNTs may be fabricated
using any suitable method. CNTs may be single-Walled carbon nanotubes (SWNTs) or multi-Walled carbon nanotubes
(MWNTs). CNT compositions that function as aAPCs include ligands attached to CNTs that bind to cell surface receptors on T cells, including at least one species of ligand
that binds to and activates the T cell receptor. T cell receptor
activators may be antigen-specific or polyclonal T cell receptor activators. Suitable antigen specific T cell receptor activators include antigens bound to MHC/HLA molecules. Exemplary antigens include, but are not limited to, viral antigens, bacterial antigens, parasite antigens, allergens and environmental antigens, tumor antigens or autoantigens. CNT
compositions that function as modular vaccines do not directly activate T cells by binding to T cell surface receptors,
but rather, facilitate the delivery of antigens to natural APCs
in vivo. Authors show that anti-CD3 antibodies adsorbed
onto SWNT bundles stimulate cells more efficiently than
equivalent concentrations of soluble anti-CD3 antibodies.
The stimulation of T-cells using anti CD3 loaded SWNT was
quantified using a mouse IL-2 ELISA. Activation with antibody inmobilized on SWNT was at least four-fold and sixfold greater in comparison to plate-bound antibodies and
soluble antibodies respectively.
The methods include isolating a population of T cells
from a subject to be treated, activating the T cells With the
CNT aAPCs, expanding the T cells, and administering the T
cells to the subject to be treated in an amount effective to
induce an immune response.

The CNT aAPCs contain antigen-presenting molecules

having determinants which match that of a selected subject
or which match any known antigen-presenting molecule determinants. The antigen-presenting molecules may be
MHC/HLA class I or class II molecules. These are used to
present antigens to T cells to activate and expand T cells
specific to the antigen. The antigen can be an allergen or
environmental antigen, such as, an antigen derived from
naturally occurring allergens such as pollen allergens, insect
allergens, animal hair and dandruff allergens and food allergens. Single walled carbon nanotubes (SWNT) were assessed for possible adjuvant properties using the model antigen ovalbumin (OVA). C57BL6 mice were treated subcutaneously with either OVA alone, with OVA adsorbed to alum,
with OVA adsorbed onto SWNT bundles, or with phosphatebuffered saline as placebo. OVA specific T cell responses
were analyzed by isolation of spleenocytes and challenged
with OVA to assess cellular immune induction by measuring
IL-2. Responses of spleenocytes from mice from all groups
were lower than SWNT OVA group including mice treated
with alum.
Other authors have presented synthetic nanocarriers for
inducing an immune response in B and/or T cells [102]. The
nanocarriers are useful for prophylaxis and/or treatment of
infectious disease, cancer, allergy, autoimmune disease and
others. Vaccines contain nanocarriers with an antigen
bounded covalent or non-covalently. The nanocarrier scaffold can be protein-based, nucleic acid based, or carbohydrate-based. Nanocarrier can be a B cell antigen and/or T cell
antigen. Authors studied in vivo T cell activation by flow
cytometry after administration of immunomodulatory
nanoparticles. Nanoparticle-encapsulated antigen generated a
more potent CD4 T cell response than free antigen in draining lymph node and distal lymph node.
This is a novel therapeutic approach to treat allergies
using immunotherapy. More studies are needed to assess
safety and effectiveness in humans but it will be a good option in the future.
There are summarized the patents of the article in the
Table 1.
CURRENT & FUTURE DEVELOPMENTS
Currently, there is definitely an urgent need for better
therapeutic approaches. Safety optimization of vaccine formulations will be one of the major issues. In addition, effective vaccines must preserve structure, reduce allergenic activity, and retain T-cell activity at the same time. Each day,
investigations show new advances in immunotherapy as
readers have seen in this article. Recently, Rask et al. [103]
published an alternative way of immunotherapy which significantly reduces the allergenicity maintaining the immunogenicity. Authors administered a lower dose of allergen with
an increased aluminum hydroxide (AlOH) concentration.
Pfaar [104] published similar results. In the other hand, some
groups of investigators study the safety/efficacy profile of
high-dose immunotherapy [105]. Soon, scientists will know
which is the way to follow in future studies of immunotherapy: more adjuvant formulation or more allergen [106].
Moreover, in these years, a number of large ongoing clinical
studies with the primary purpose of establishing the EMA

32 Recent Patents on Inflammation & Allergy Drug Discovery 2014, Vol. 8, No. 1

Table 1.

El-Qutob et al.

Summary of Patents.

Allergen-Specific Patents
Hypoallergenic polypeptides for the treatment of house dust mite allergy

Two mosaic Der p 1 and Der p 2 vaccines for immunotherapy of HDM

allergy

Recombinant Der p 2 expressed in Pichia pastoris as a natural-like allergen for immunotherapy and diagnostic purposes

Recombinant Der p 2 for therapeutic or diagnostic use

Contiguous overlapping peptides (COPs) for treatment of birch pollen allergy

COPs fragments provided from birch pollen Bet v 1

Hybrid proteins from Parietaria judaica major allergen and uses thereof

Dimeric hypoallergenic proteins of Parietaria judaica and their use for

immunotherapy

Vaccine peptide combinations against cat allergy

Polypeptide combinatios of Fel d1 protein fragments which bind to many

different MHC Class II molecules

Vaccine comprising Amb a 1 peptides for use in the treatment of ragweed

allergy

Combinations of peptide fragments derived from the major allergen in

ragweed pollen, Amb a 1

Novel therapies in grass pollen allergies

Use of polymerized polypeptides in grass pollen allergic patients

Non-Allergen-Specific Patents
Use of an adjuvanted allergy vaccine formulation for parenteral administration

Up-dosing phase of immunotherapy is initiated after start of the grass

pollen season

Hypallergenic mosaic antigens and methods of making same

Cleaving a natural allergen in non-overlapping peptide fragments and

recombining them in different order

An allergen dosage form

A pharmaceutical product suitable for oromucosal administration of an

allergen comprising at least one allergen and a matrix in form of a fastdispersing non-compressed solid dosage form

Allergoid derived from allergens

Reduction of allergenicity of allergens characterized in that all or a part of

the primary amine groups of the lysine and arginine residues are modified
with carbamylation or thiocarbamylation reaction, and reaction with a
dialdehyde or diketone

Preparations of conjugates comprising adenine derivatives and allergenic

proteins and their use for specific immunotherapy of allergenic diseases

Preparation of stable conjugates between adenine derivative and allergenic

proteins

Suppression of a hypersensitivity immune response with unrelated antigen

derived from allergen source material

Treatment of a hypersensitivity immune response in an individual with an

antigen unrelated to the allergen triggering a hypersensitivity immune
response in the individual

Allergen peptide fragments and use thereof for treatment of dust mite allergies

COPs fragments which together comprise the entire amino acid sequence of
an allergen

Carbon nanotube compositions and methods of use thereof

Use of carbon nanotubes with antigens for immunotherapy. Use of carbon

nanotubes as adjuvant

Vaccine nanotechnology

Use of nanoparticle for T cell stimule

(European Medicines Agency) guideline documentation

needed for registration of pharmaceutical products will certainly add further to our basic knowledge about the potential
immunological modifications and long-term options. This is
of great importance for the optimization of the future treatment of the increasing number of patients suffering from
respiratory allergic diseases.

ACKNOWLEDGEMENTS
Declared none.
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