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\begin{document}

\title{On the role of antibody affinity in the IgE mediated allergic response}



\author[1]{Sviatlana Starchenka}%
\author[2]{Martin Bachmann}%
\author[2]{Pascal S. Krenger}%
\author[2]{Mona Mohsen}%
\author[1]{Matthias Kramer}%
\author[3]{Piers Whitehead}%
\author[2]{Monique Vogel}%
\author[1]{Matthew Heath}%
\affil[1]{Allergy Therapeutics Plc}%
\affil[2]{University Clinic for Rheumatology and Immunology Inselhospital}%
\affil[3]{SeromYx Systems}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Type I hypersensitivity, also known as classical allergy, is mediated
via allergen-specific IgE antibodies bound to type I FcR (Fc\selectlanguage{greek}ε\selectlanguage{english}RI) on the
surface of mast cells and basophils upon cross-linking by allergens.
This IgE-mediated cellular activation may be blocked by
allergen-specific IgG through multiple mechanisms, including direct
neutralization of the allergen or engagement of the inhibitory receptor
Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb which blocks IgE signal transduction. In addition, co-engagement
of Fc\selectlanguage{greek}ε\selectlanguage{english}RI and Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb by IgE-IgG-allergen immune-complexes causes
down-regulation of receptor bound IgE, resulting in desensitization of
the cells. Both, activation of Fc\selectlanguage{greek}ε\selectlanguage{english}RI by allergen-specific IgE and
engagement of Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb by allergen-specific IgG are driven by
allergen-binding. Here we delineate the distinct roles of antibody
affinity versus avidity in driving these processes and discuss the role
of IgG subclasses in inhibiting basophil and mast cell activation.%
\end{abstract}%



\sloppy


\textbf{On the role of antibody affinity in the IgE mediated allergic
response}

Martin F. Bachmann\textsuperscript{1,2,3*}, Pascal S.
Krenger\textsuperscript{1,2}, Mona O. Mohsen\textsuperscript{1,2},
Matthias F. Kramer\textsuperscript{4,5}, Sviatlana
Starchenka\textsuperscript{4}, Piers Whitehead\textsuperscript{6},
Monique Vogel\textsuperscript{1,2} \textsubscript{,} and Matthew D.
Heath\textsuperscript{4}

\textsuperscript{1}Department of Immunology, University Clinic for
Rheumatology and Immunology, Inselhospital, Bern,
Switzerland;\textsuperscript{2}Department of Biomedical Research Bern
(DBMR), University of Bern, Bern, Switzerland;
\textsuperscript{3}Nuffield Department of Medicine, The Jenner
Institute, University of Oxford, Oxford, UK; \textsuperscript{4}Allergy
Therapeutics, Worthing, UK;\textsuperscript{5}Bencard Allergie GmbH,
Munich, Germany;\textsuperscript{6}SeromYx Systems, Cambridge, United
States.

*Corresponding author: Martin F Bachmann\textbf{,} Department of
Immunology, University Clinic for Rheumatology and Immunology,
Inselspital, Bern, Switzerland. E-mail: martin.bachmann@me.com

\textbf{ORCID ID}

Martin F. Bachman -- https://orcid.org/0000-0003-4370-2099

Pascal S. Krenger -- https://orcid.org/0009-0008-7074-3842

Mona O. Mohsen -- https://orcid.org/0000-0003-3510-9148

Matthias F. Kramer -- https://orcid.org/0000-0002-3740-4733

Sviatlana Starchenka -- https://orcid.org/0000-0002-4914-4051

Piers Whitehead -- Not available

Monique Vogel -- https://orcid.org/0000-0002-5219-4033

Matthew D. Heath -- https://orcid.org/0000-0002-6095-4098



\textbf{Abstract}

Type I hypersensitivity, also known as classical allergy, is mediated
via allergen-specific IgE antibodies bound to type I FcR (Fc\selectlanguage{greek}ε\selectlanguage{english}RI) on the
surface of mast cells and basophils upon cross-linking by allergens.
This IgE-mediated cellular activation may be blocked by
allergen-specific IgG through multiple mechanisms, including direct
neutralization of the allergen or engagement of the inhibitory receptor
Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb which blocks IgE signal transduction. In addition, co-engagement
of Fc\selectlanguage{greek}ε\selectlanguage{english}RI and Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb by IgE-IgG-allergen immune-complexes causes
down-regulation of receptor bound IgE, resulting in desensitization of
the cells. Both, activation of Fc\selectlanguage{greek}ε\selectlanguage{english}RI by allergen-specific IgE and
engagement of Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb by allergen-specific IgG are driven by
allergen-binding. Here we delineate the distinct roles of antibody
affinity versus avidity in driving these processes and discuss the role
of IgG subclasses in inhibiting basophil and mast cell activation.

\section*{KEYWORDS}

{\label{keywords}}

Affinity, allergy, antibody, avidity, IgE.\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Future Research Perspectives} Specific immunotherapy should aim
at maximizing allergen-specific IgG responses. IgG subclass responses
after AIT are not fully understood to date and require ongoing clinical
research, further development of platforms e.g. Immunocap\selectlanguage{ngerman}® for IgG1
could provide high throughput value. Further study on the
characterization of Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb on human mast cells may further establish
evidence of the translational potential of vaccines containing a single
B-cell epitope, which may be able to block some of the specific IgG and
block mast cell activation additionally by engaging FcgRIIb. Developing
adjuvants that promote B cell responses represents a major challenge in
vaccinology today and is relevant for the development of next generation
allergy immunotherapies.\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Major milestone discoveries} High avidity interactions, and
consequently, low affinity IgE antibodies can trigger cellular
activation. Antibody affinity dictates the mechanism of mast cell
inhibition, and low affinity IgG antibodies triggering the inhibitory
Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb pathway can show a broader cross-reactivity pattern than
previously thought. IgG subclasses plays only a minor role in inhibiting
the allergic response; IgG1 as an equally dominant IgE-blocking antibody
as IgG4. Repetitive display of allergens on VLPs packaged with TLR
ligands appears an attractive way to broaden and increase induction and
maintenance of specific antibodies; the basis of a novel vaccine against
peanut allergy.\tabularnewline
\bottomrule
\end{longtable}

\textbf{Glossary}

\textbf{Allergen-specific immunotherapy (AIT):} also known as
desensitization or hyposensitization; the most common form of specific
immunotherapy; involves a course of injections that build up tolerance
to particular allergens through small, controlled
doses.\textbf{Allergens:} proteins that trigger the immune system of an
allergic person to produce unwanted symptoms. \textbf{Allergy:} a
chronic condition involving an exaggerated reaction to an otherwise
harmless substance, called an allergen. \textbf{B cells:} differentiate
into clones of antibody-producing plasma cells; have a central role in
allergen tolerance through the production of IgG-blocking
antibodies.\selectlanguage{romanian}\textbf{FcƐRI:} a high-affinity IgE receptor expressed on the
cell surface of mast cells and basophils; triggers the IgE-mediated
allergic cascade. \selectlanguage{greek}\textbf{FcγRIIb:}\selectlanguage{english} a low-affinity IgG receptor. IgG
antibodies can inhibit IgE-mediated mast cell activation through direct
allergen neutralization or through the inhibitory receptor
Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb.\textbf{Germinal center:} a specialized microstructure that
forms in secondary lymphoid tissues, producing long-lived
antibody-secreting plasma cells and memory B cells, which can provide
protection against reinfection; \textbf{IgE:} primary antibody mediator
in the allergic response. \textbf{IgG:} most common antibody in the
body; used to treat a range of diseases. \textbf{Mast cells:} similar to
basophils, mast cells become activated by antigen crosslinking of Fc\selectlanguage{romanian}ƐRI
receptor-bound IgE to undergo rapid degranulation and release of the
inflammatory substance histamine, causing an allergic
reaction.\textbf{Pathogen-associated structural pattern (PASP):} a
pathogen-associated molecular pattern (PAMP). Cells of the innate immune
system utilize pattern recognition receptors to identify viral pathogens
by engaging PAMPs e.g. viral structures, Toll-like receptor ligands such
as RNA. \textbf{Toll-like receptors:} The Toll-like receptors are
Pathogen recognition receptors that have a unique and essential function
in animal immunity.

\textbf{1} \textbf{Introduction}

The discovery of IgE-mediated allergic disease as an indication of
hay-fever, or allergic rhinitis, was first described in 1819 by John
Bostock, a medical doctor who himself suffered from hay
fever\textsuperscript{1}. He called the disease catarrhus aestivus,
summer catarrh, as it was invariably absent in winter time. Catarrhus
aestivus was a rare disease in the early nineteenth century, and after
reporting his own case, it took him more than nine years to identify
another 28 cases for publishing a second article\textsuperscript{2}. At
the beginning of the 20\textsuperscript{th} century, allergens were
considered being toxins\textsuperscript{3}. Only with the discovery of
IgE antibodies\textsuperscript{4,5}, it became evident that
allergen-specific antibodies are the reason for the aberrant response of
the body against pollen.

In clinical practice, there are several possibilities to avoid the
activity of IgE and many pharmaceutical interventions aim to prevent IgE
production and/or make the immune system more tolerant to allergens by
inducing a shift in Th cell responses from Th2 to Th1 or regulatory T
cells\textsuperscript{6,7}; more recently, the concept of mAbs targeting
and blocking IgE directly by monoclonal anti-IgE antibodies has been
introduced\textsuperscript{8}. Alternatively, it may be possible to
block the action of IgE indirecty by induction of allergen-specific IgG
through passive\textsuperscript{9} or active
vaccination\textsuperscript{10,11}. This approach is supported by the
clinical observation that successful specific immunotherapy may
correlate with an increase of the IgG/IgE ratio, and more recently, that
polyclonal and monoclonal allergen specific IgG antibodies were able to
curb allergic immune responses both in mice and humans as a form of
passive vaccination\textsuperscript{12-14}. Hence, it seems reasonable
to conclude that the overarching goal of immunotherapy should be the
induction of allergen-specific IgG antibodies.

This raises the question of antibody specificity and quality. Which are
the key parameters driving or inhibiting allergic responses? As outlined
below, the answer is complex and different for IgE mediated activation
by allergen versus IgG mediated neutralization of allergen versus
Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb-mediated inhibition of allergic responses (Figure 1). The same
is true for antibody specificity, as rules for IgE-mediated activation
versus IgG-mediated blockade or Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb-mediated inhibition are
fundamentally different. Perhaps unexpectedly, IgG subclasses plays only
a minor role in inhibiting the allergic response.

\textbf{2 Low affinity IgE antibodies suffice to drive the allergic
responses: avidity provides the key}

Antibody affinity, also known as binding affinity and defined by its
equilibrium constant, is the strength of the interaction between the
antigen-binding site on an antibody and a specific epitope on an antigen
(monovalent binding) and can be defined by its equilibrium constant
(Figure 2A). Antibody avidity represents the overall strength of the
antibody-antigen interactions and is influenced by several factors in
particular multivalent binding (Figure 2A). In essence, the greater an
immunoglobulin's valency (number of antigen binding sites), the greater
its potential avidity as it can bind multiple epitopes on a single
antigen -- provided the antigen is multivalent\textsuperscript{15}.

Most classical physico-chemical analysis is performed in solution, in
part to avoid the effects of multivalent binding. Nevertheless, standard
antibody binding assays, such as ELISA, surface plasmon resonance, and
Biolayer Interferometry are performed with antibodies in solution; their
ligands are, however, typically bound to two-dimensional surfaces. This
opens the possibility for antibodies to bind their ligands with 2 arms,
a problem that is typically avoided by complicated coating techniques,
including coating at low density to avoid avidity effects (caused by
multivalent binding), as affinity (referring to monovalent binding) is
much better defined by classical binding models than avidity. However, a
notion often overseen is that most pathogens i) are recognized on 2
dimensional surfaces and ii) are highly polyvalent and therefore prone
to bind antibodies in a multivalent fashion. Indeed, natural IgM
antibodies bind viral particles with great efficiency due to decavalent
binding which makes many antibodies stick like glue even if the binding
affinity of an individual variable region is low and barely
measurable\textsuperscript{16}. Hence, avidity may be more important in
a real-life setting. In the case of IgE bound to Fc\selectlanguage{greek}ε\selectlanguage{english}RI on the surface of
effector cells, nature adds one level of complexity: lateral diffusion.
While in ELISA plates, ligands are fixed on plastic, IgE molecules bound
to Fc\selectlanguage{greek}ε\selectlanguage{english}RI can rapidly diffuse along the cell surface membrane, a process
that strongly facilitates multivalent, high-avidity binding of IgE
antibodies to allergens (Figure 2B). In this context, it is interesting
to note that the restriction of the movement of receptor-bound IgE
within the 2 dimensions of the cell membrane results in high local
concentrations of the IgE molecules as well as allergens bound to IgE.
Indeed, 10'000 molecules bound to the cell surface of regular sized
cells may exhibit a local concentration of
\textgreater{}10\textsuperscript{-6} M within the
membrane\textsuperscript{17}. In addition, membrane bound molecules
diffuse on the cell surface with a high velocity, allowing them to
circle the cell once every second\textsuperscript{18}. Hence all these
properties foster multivalent binding of single allergens to membrane
bound IgE recognizing different epitopes on the allergen. Indeed, we
could show that IgE antibodies exhibiting an affinity as low as
10\textsuperscript{-6}M for the allergen Fel d 1, were nevertheless able
to bind multivalently to Fc\selectlanguage{greek}ε\selectlanguage{english}RI bound IgE on mast cells and cause
degranulation of the cells\textsuperscript{19}. This was not possible if
the membrane was brought below the Krafft point, the temperature at
which cholesterol in membranes freezes, not allowing for lateral
diffusion of molecules (Figure 2C). Thus, lateral diffusion of IgE
allows for multivalent binding of allergen, causing high avidity
interactions, and consequently, low affinity IgE antibodies can trigger
cellular activation. This effect may be particularly pronounced for
dimeric allergens. Indeed, a case in point is the cat allergen Fel d 1,
which is naturally dimeric and causes particularly strong allergies.
These data may also explain the often unexpectedly high cross-reactivity
between structurally unrelated allergens such as birch allergens and
latex.

\textbf{3 High affinity antibodies are required for
allergen-neutralization}

The most straightforward way for IgG antibodies to inhibit activation of
basophils and mast cells by allergens is direct neutralization of the
allergen in competition with allergen-specific
IgE\textsuperscript{10,20}. This mechanism, now, corresponds to the
above discussed and by physico-chemists' preferred interaction of
antibodies with ligands; it happens in solution, or at least within
tissue fluids and antibodies typically bind with one arm only. Hence,
because this epitope-specific interaction is a competitive one, it is
largely promoted by high affinity and monovalency, in contrast to
avidity which only plays a minor role in direct neutralization. Indeed,
low affinity IgG antibodies failed to neutralize allergens and did not
block basophil/mast cell activation both \emph{in vitro} as well as
\emph{in vivo}\textsuperscript{21}. As a further restriction,
neutralizing antibodies must recognize the same allergen as the IgE and
preferably even the same epitope. This contrasts with interaction
mediated via Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb\emph{,} as described below. Indeed, low affinity
IgG antibodies fail to neutralize the allergen but may efficiently block
cellular activation by engaging Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb.

\selectlanguage{greek}\textbf{4 Low affinity antibodies suffice to engage FcγRIIb: abrogating
IgE signalling for single allergens}\selectlanguage{english}

Using Fel d 1 as a model antigen, we have shown that low affinity IgG
antibodies fail to neutralize the allergen but nevertheless can
efficiently block mast cell/basophil activation\textsuperscript{21,22}.
This unexpected finding was entirely Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb dependent. Furthermore, the
low affinity antibodies must have a different epitope specificity than
the IgE, as only in this case, low affinity IgG could block mast cell
activation by interaction \emph{with} Fc\selectlanguage{greek}ε\selectlanguage{english}RI-bound IgE and engagement of
the inhibitory Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb. Again, it is likely that lateral diffusion and
avidity stabilization is important in the process, as low affinity IgG
may need to rapidly engage with and be stabilized by Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb for
effective inhibition of cellular activation. Thus, in this way, also low
affinity IgG antibodies can ``poison'' IgE-signalling by engaging
Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb. There is an interesting analogy from enzyme kinetics for the
two types of inhibition. Allergen neutralization corresponds to
competitive inhibition, requiring high affinity ligands for effective
competition. In contrast, engagement of Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb corresponds to
con-competitive inhibition, rather independent of high
affinity\textsuperscript{23}.

\selectlanguage{greek}\textbf{5 FcγRIIb shows inhibitory activity across epitopes and
allergens in allergen mixtures: abrogating IgE signalling for multiple
allergens}\selectlanguage{english}

A key difference between allergen neutralization and engagement of
Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb is the breadth of activities, not only in terms of affinity but
also in terms of specificity. As discussed above, neutralization is only
possible for a given epitope on a given allergen. Unexpectedly,
engagement of Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb not only blocks activation of IgE antibodies with
a different epitope than the IgG exhibits but is also effective for
entirely different allergens as long as the allergen recognized by the
IgG antibody is present in the mixture. Specifically, peanut-allergic
local and systemic responses against whole peanut allergen extract could
be blocked by immunization against a single allergen, specifically Ara
h1 or Ara h2\textsuperscript{13,24}. Even though mice were allergic to
multiple peanut allergens, immunization against a single allergen was
sufficient to block allergic responses. Furthermore, polyclonal
antibodies against Ara h 1 or Ara h 2, as well as a mAb against Ara h 2
were able to abrogate allergic responses induced by the peanut
extract\textsuperscript{25}, confirming earlier data for cat allergy, an
allergy which is, however, mostly driven by the single allergen Fel d
1\textsuperscript{26}. This protective effect of Ara h 1 and Ara h 2
antibodies was again strictly dependent on the presence of functional
Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb. In absence of the inhibitory receptor, no reduction in allergic
symptoms could be observed. Hence, IgG complexes with a single allergen
are able to abrogate IgE-signalling induced by complex allergen
mixtures.

\textbf{6 Role of the IgG subclass}

Clinical efficacy of allergen-specific immunotherapy (AIT) or
vaccination best correlates with increased levels of IgG4 in humans.
Hence, it is generally assumed that IgG4 is the key IgG subclass for
clinical outcomes. However, as pointed out
previously\textsuperscript{27}, the correlation between clinical
efficacy and IgG4 may simply be due to the fact that the way we are
currently performing immunotherapy is preferentially inducing IgG4
rather than other subclasses; mostly due to the absence of strong
toll-like receptor or other innate ligands in the formulations. It
should be noted that a correlation between clinical outcome and
allergen-specific IgG4 has been reported in some but not all studies.
Hence, IgG4 is not a validated biomarker\textsuperscript{28}. Negative
results may in part be due to suboptimal designs of conventional
immunotherapy. Recently, however, it was demonstrated that IgG1 and IgG4
appeared in mucosal fluids after AIT with genetically modified
allergens\textsuperscript{29}. IgG subclass responses after to AIT are
not fully understood to date and are therefore an active area of ongoing
clinical research.

We compared the ability of 3 different mAbs against Fel d 1 expressed in
a IgG1 or IgG4 format (exhibiting identical epitope specificities
despite being expressed as IgG1 or IgG4) for their ability to block
primary human basophil activation via allergen-neutralization or
engagement of Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb\textsuperscript{27}. Indeed, both antibody
subclasses had the same ability to block basophil activation in a
quantitative manner. These observations were confirmed when the affinity
of the antibodies for recombinant Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb was assessed by Biolayer
Interferometry showing similar affinities for IgG1 and IgG4. Hence, it
seems that different IgG subclasses -- at least IgG1 and IgG4 -- are
capable of similar allergen neutralization and Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb engagement. It is
now planned to further assess the diversity and functionality of
antibody profiles in response to a vaccine against peanut allergy which
is in clinical development (PROTECT trial; ClinicalTrials.gov
Identifier: NCT05476497).

\textbf{7 Current trends in AIT}

Current trends in AIT seek novel routes of allergen administration as
e.g. the use of allergens or allergoids formulated with adjuvant systems
or native allergens displayed on virus-like particles (VLPs). Many of
these novel approaches favour induction of IgG1 rather than
IgG4\textsuperscript{14}. It could therefore be considered that
allergoids either share some structural features with viral antigens as
eg forming aggregates that present the epitopes in a more rigid and
repetitive structures compared to native allergens. Ordered and
repetitive surfaces are unique features of viral and bacterial surfaces
and, hence, considered to be a pathogen-associated structural pattern
(PASP)\textsuperscript{30}.

Such considerations may offer insights into the surprisingly large
treatment effect size in a recent exploratory field trial using a mixed
Grass-SCIT-based allergoid combined with a novel adjuvant system
(MCT\selectlanguage{ngerman}®-MPL®) in the late stages of clinical
development\textsuperscript{31}. Approximately a 40\% reduction of the
Combined Symptom and Medication Score were achieved during the peak
Grass Pollen Season (GPS) compared to placebo using a new regimen of
only six pre-seasonal monthly injections\textsuperscript{31}. In this
study, grass-specific serum of IgG4 (n=37) and total IgG (n=10) was
statistically significantly increased (LS mean ± SE: +3.34 mg/L ±0.946,
p = .0006 and 79.94 mg/L ±111.10 (p = .0004)) at the start of the grass
pollen season and remained elevated at the end of the grass pollen
season.

It is worth noting that detection of specific IgG1 antibodies is not
possible with the high throughput ImmunoCap Phadia platform used to
support large-scale sampling from clinical trials. However, a grass
specific (mix) IgG is commercially available and covers all subclasses
including IgG1 and IgG4. Based on the fact that IgG1 makes up 60\% of
all IgG subclasses while IgG4 only 4\%, measurement of grass specific
total IgG will likely mainly represent changes in IgG1 levels.

Given recent developments in the field studying the importance of IgG
subclasses and the classification of IgG1 as an equally dominant
IgE-blocking antibody as IgG4, this will be a further potential
biomarker correlate of the pivotal phase III trial, where specific IgG1
data is currently being collected for a sub-population-group of patients
on active treatment. This will add further clinical evidence whether
IgG1 and IgG4 subclass induction are equally important and both may
constitute a hallmark of successful short-course pre-seasonal AIT;
attributed to its adjuvant system (including a toll-like receptor 4
ligand) and nature of ligands (allergoid as opposed to native allergens)
in the formulation.

\textbf{8 TLR7/8 signalling broadens antibody repertoire, affinity, and
avidity}

Despite the above-described ability of low affinity allergen-specific
IgG antibodies to block type I hypersensitivity responses, it remains
common thinking that high affinity antibodies would be more effective to
also neutralize the allergen. In addition, induction of high affinity
antibodies continues to be a general and common goal in vaccinology. For
that, induction of germinal centers (GC), also known as secondary
follicles, is a key goal, as GCs are the anatomical location of
hypermutation and antibody affinity maturation. Induction of GCs is
driven by antigen deposition on follicular dendritic cells (FDCs) and
effective stimulation of B cells. Repetitive display of antigens in
general and allergens specifically has been found to induce strong and
rapid B cell responses due to effective B cell receptor cross-linking as
well as engagement of the innate immune system\textsuperscript{30}.
Indeed, natural IgM recognizes repetitive epitopes on viruses and
virus-like particles (VLPs), causing activation of the classical pathway
of complement (via C1q), leading to the deposition of these particles on
FDCs and to GC-reactions \textsuperscript{16}. In addition, VLPs may
package RNA from \emph{E. coli} during VLP production, when assembling
inside bacteria. This RNA has not only been observed to effectively
drive class switching to IgG and IgA but also to increase the affinity
of the induced antibodies as well as to facilitate maintenance of a
broad immunoglobulin repertoire against both VLPs as well as displayed
allergens\textsuperscript{32}. In a recent preclinical model of peanut
allergy, the presence of RNA in VLPs (Cucumber mosaic virus-like
particle; CuMV\textsubscript{TT}) displaying the peanut allergen Ara h 2
(CuMV\textsubscript{TT}-Ara h 2; VLP Peanut) proved essential for
induction of protective IgG responses against peanut allergy; here the
role of TLR-signalling and its influence on the vaccine's efficacy
profile was explored in TLR7 knock out (KO) mice as well as VLPs loaded
with low amounts of RNA\textsuperscript{33}. Unexpectedly the total
amount of Ara h 2 specific antibodies was not affected by TLR 7
signalling; in contrast, when mice were immunized with the product
containing approximately half the total RNA content, a significant
reduction of Ara h 2 specific IgG titers was observed in Wild Type and
TLR 7 KO mice. Not only the total amount of IgG antibodies was affected
but also IgG subclasses and the number of high avidity IgG antibodies
(Figure 3). In essence, Ara h 2 specific total IgG responses were highly
dependent on the VLP Peanut carried RNA. The observed difference in low
RNA versus lack of TLR 7/8 is explained by the fact that the RNA loaded
in VLPs also engages TLR 3, contributing to the overall immunogenicity
profile. Indeed, the bacterial RNA contained in VLP Peanut exists in
both single- and double-stranded forms, stimulating TLR 7 and TLR 3. A
missing TLR 7 signal can therefore partially be compensated by a TLR
3-derived signal as both TLRs are expressed in B
cells\textsuperscript{34,35}.

Additionally, there seems to be a different dependency on the
VLP-carried RNA between antigen- and carrier-specific immune responses.
The observation that VLP-specific antibodies were less dependent on TLR
signalling than Ara h 2 specific antibodies may be explained by the fact
that the CuMV\textsubscript{TT} subunits are more densely packed at a
lower distance than the Ara h 2 molecules, overcoming TLR dependence.
This indicates that the overall IgG response is dictated by a
2-dimensional integral of TLR stimulation and antigen density. We have
previously seen that B cell activation may be driven by integrated
overall signals, as TLR-signalling could overcome IL-21-dependence of B
cell responses\textsuperscript{36}; an observation reminiscent of the
observed independent of TLR-signalling.

Hence, repetitive display of allergens on VLPs packaged with RNA appears
an attractive way to increase induction and maintenance of high affinity
antibodies (Figure 4). In studies on mast cell interaction with VLPs
displaying Fel d 1 or peanut allergens, a substantially reduced
interaction with IgE bound to mast cells was observed and an even more
pronounced failure to significantly activate the Fc\selectlanguage{greek}ε\selectlanguage{english}RI mediated
signalling cascade. Specifically, under conditions where similar or
higher amounts of allergen were bound to IgE on mast cells, free
allergens induced strong cellular activation while allergens on VLPs
failed to do so\textsuperscript{13,14}. Together, it was therefore an
attractive choice to bring Ara h 2 displayed on VLPs (VLP Peanut) into
clinical development\textsuperscript{24}.

\textbf{9 Conclusions}

The affinity as well as the avidity of IgE-allergen interactions play
critical roles for activation of effector cells and allergic reactions.
It is well accepted that specific allergen-binding of antibodies is
required for cellular activation. However, the low affinity of
Fc\selectlanguage{greek}ε\selectlanguage{english}RI-bound IgE for allergens can be compensated by bivalent, high
avidity binding to multiple epitopes on allergens. This appears to be
the mechanism responsible for the often not expected allergen
cross-reactivity which may be driven by low affinity but high avidity
interactions. Similarly, IgG antibodies with low affinity for the
allergen were also sufficient to inhibit mast cell activation by
engaging the inhibitory Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb. Furthermore, polyclonal and monoclonal
antibodies specific for a single allergen were able to block allergic
symptoms mediated by whole allergen extract. This effect was also
mediated by Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb and was rather independent of the affinity of the
antibodies for the allergen as low affinity antibodies were also
effective. Interestingly, comparison of two different IgG subclasses,
IgG1 and IgG4 showed the same capacity to block mast cell activation and
to bind to Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb, suggesting similar clinical efficacy for both
antibody subclasses. This is currently being further validated in the
clinical setting for short-course allergoid treatment options which
utilizes an adjuvant system employing MPL (TLR 4 agonist).

Finally, even if Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb-mediated inhibition can be triggered by low
affinity IgG antibodies, allergen neutralization still requires high
affinity antibodies which are generated in the germinal centers. The
generation of such antibody responses has been shown to be optimized by
repetitive display and TLR signalling in B cells, which can be triggered
by immunization with virus-like particles packaged with RNA and
displaying antigens in a repetitive fashion.

\textbf{Acknowledgments}

We thank Daniel Speiser for the critical review of the manuscript. The
figures were created with BioRender.com.

\textbf{Funding}

This work is supported by the Swiss National Science Foundation (SNSF)
(grant Nr. 310039\_185114) and DeepVax GmbH.

\textbf{Author contributions}

M.F. Bachmann, P.S. Krenger, M.O. Mohsen, M.F. Kramer, S. Starchenka, M.
Vogel, P. Whitehead, M.D. Heath wrote, revised, and approved this
manuscript.

\textbf{Conflict of Interests}

M.F. Bachmann, M.O. Mohsen, M. F. Kramer, S. Starchenka, M.D. Heath are
involved in the development of allergen-specific immunotherapies within
Allergy Therapeutic LTD and Saiba AG.

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\textbf{FIGURES}

\textbf{Figure 1.} \textbf{Model illustrating the impact of IgG
antibodies on Type I hypersensitivity} . Allergens are taken up by
antigen presenting cell which present processed antigen to
T\textsubscript{H} cells. T\textsubscript{H} cells help B cells to
produce IgE which binds to Fc\selectlanguage{greek}ε\selectlanguage{english}RI on the surface of mast cell and
sensitize them. Allergen-specific IgG antibodies neutralize allergens
and/or engage the inhibitory receptor Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb thereby agonist mast cell
activation.

\textbf{Figure 2.} \textbf{Strength of interaction between antibody and
antigen.} A) Difference between antibody affinity and avidity. B-C)
Lateral movement at 37\selectlanguage{ngerman}°C facilitates multivalent and high-avidity
binding of IgE antibodies to allergens, while temperatures below the
Krafft point at (e.g. 4°C) inhibit lateral movement and multivalent
binding.

\textbf{Figure 3. The RNA dependency of VLP Peanut to induce
high-avidity antibodies and to drive antibody class switching.} A)
Illustration of VLP Peanut (PDB file). VLP Peanut consists of
CuMV-derived subunits (PDB code 1F15) and CuMV subunits that were
modified by the incorporation of Ara h 2 (PDB code 3OB4). For the
illustration, the Ara h 2 monomer was manually placed near the insertion
loop present in the CuMV subunit by using COOT. Based on the obtained
modified PDB file and icosahedral symmetry operators the image was
created in pymol. The figure was curtosy of Prof Kaspars Tars, Riga B)
Cartoon representation of VLP Peanut encapsulated RNA. C) Ara h 2
specific total IgG responses are highly dependent on the VLP
Peanut-carried RNA. Illustrations depicted in B and C created with
Biorender.com.

\textbf{Figure 4.} \textbf{A cartoon illustrating a VLP
nanoparticle-based vaccine against peanut allergy.} The nanoparticles
are packed with ssRNA, a TLR7/8 ligand, and display Ara h2 antigen. The
natural pentameric IgM antibody can bind the VLPs with low affinity/high
avidity. Such interaction contributes to the activation of classical
complement cascade and binding of the formed immune-complex on FDCs for
effective formation of GCs. This vaccine may induce high-affinity
antibodies, drive antibody class-switching and establish long-lived
memory. Abs; antibodies.\selectlanguage{english}
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