Mouse models of peanut hypersensitivity:
optimzation for future studies of immunotherapy with nanoparticle adjuvants
Jessica A. Jerez
Project Director: Carlos Gamazo, PhD
Background: Peanut-induced anaphylaxis (PIA) represents an
important public health problem and can be life-threatening for patients.
Oral immunotherapy (OIT) may be an effective tool for desensitization,
and nanoparticle adjuvants may enhance OIT results, but investigation
is currently in the preclinical stage. To date, numerous murine models
have been studied, though PIA has been difficult to induce and results
difficult to reproduce.
Objectives: To optimize a murine model of peanutinduced anaphylaxis, selecting a mouse strain as well as
sensitization and antigen challenge protocols that more
realistically parallel the development of PIA in humans. To
use the selected mouse model in studies that test the
effectiveness of nanoparticle vaccines as OIT against PIA.
Strain selection: C57BL/6 vs ICR
IgG1 binding in mice sensitized to
peanut (sera diluted 1/160)
Methods: Female C57BL/6 and ICR mice were subjected
to oral sensitization with peanut proteins over a period of 4
weeks, with naïve mice as negative controls. 2 weeks later,
mice were challenged with peanut extract via intraperitoneal
(i.p) injection and monitored for signs of anaphylaxis and
temperature decrease. Blood and faeces were collected
throughout for analysis of peanut-specific IgG1, IgG2a, IgE,
and IgA.
IgG1 binding in mice sensitized to
peanut (sera diluted 1/5120)
1.8
1.4
1.6
ICR
ICR
1.35
1.05
C57
0.45
ICR
1.2
OD
0.9
OD
OD
IgG2a binding in mice sensitized to
peanut (sera diluted 1/40)
0.7
0.35
0.8
C57
0.4
C57
0
0
1
2
3
4
5
0
6
0
1
Time after initial sensitization (weeks)
2
3
4
5
0
6
0
1
Time after initial sensitization (weeks)
2
3
4
5
6
Time after initial sensitization (weeks)
Figure 2. Peanut-specific antibody levels in C57 and ICR mice. ICR mice display a more imbalanced TH2-response than C57 mice. Samples consisted of pooled aliquots from individuals within the same protocol group
(i.e. sensitized ICR vs sensitized C57 mice). A) For C57 mice, IgG1 levels can be seen to steadily increase as expected. ICR samples at this dilution (1/160) lead to signal saturation, seen as a sharp rise followed by a
plateau. B) With sera diluted to 1/5120, the steadily rising level of peanut-specific IgG1 in ICR mice can be seen more clearly. C) Following sensitization, peanut-specific IgG2a levels are greater in ICR mice than C57 mice.
d:
0
7
14
21
28
35
42
Progressive temperature changes in mice
after challenge with peanut extract
Fig. 1. Sensitization and challenge schedule.
Mean temperature change following challenge with
peanut extract: complete vs hydrophilic fraction
1.5
40
0
C57, sensitized (n=14)
0
ICR, naïve (n=3)
ICR Naïve, Complete
PE (control)
1
0
ICR, sensitized (total)
46.2
7
hydrophilic PE (n=7)
42.9
complete PE (n=6)
50
0.75
38.5
Naive, Complete
Naive, Hydrophilic
C57: Sensitized, Complete
Sensitized, Complete
Sensitized, Hydrophilic
ICR Sensitized,
Complete PE
37
ICR Naïve controls:
hydrophilic or complete PE
ICR Sensitized,
Hydrophilic PE
35.5
ICR Sensitized,
Hydrophilic PE
0
-0.75
-1.5
Naive, Hydrophilic
Naive, Complete
Sensitized, Hydrophilic
Sensitized, Complete
C57 Sensitized, Complete
-2.25
34
10
20
30
40
-3
Time after challenge administration (min)
Results: Sensitized ICR mice exhibited higher peanut-specific
antibody levels (particularly IgG1) and greater incidence of severe
anaphylactic reactions than sensitized C57 mice.
Challenge protocol
Figure 3. Changes in rectal temperature over time due to challenge with peanut extract (either complete or hydrophilic fraction). A) Sensitized ICR mice exhibited greatest temperature drops. All lines indicate trends for ICR
groups unless otherwise indicated. Each point represents the protocol group average. B) Among ICR mice, greater temperature drops were seen in sensitized vs naïve mice (p = 0.018), but there was no significant difference between
hydrophilic and complete peanut extract (p=0.53).
Sensitization and challenge routes
IgG1 levels in ICR mice: effect of
sensitization and challenge protocols
1.1
IgG2a levels in ICR mice: effect of
sensitization and challenge protocols
0.6
Double sensitization, oral or IP challenge
0.3
OD
0.825
0.55
Oral + EC (S), IP (Ch)
Oral + EC (S), oral (Ch)
Oral (S), oral (Ch)
Oral (S), IP (Ch)
Oral placebo, IP (Ch)
Double placebo, IP (Ch)
0.275
0
IgE levels in mice sensitized to peanut
proteins
0.4
Double sensitization,
oral or IP challenge
OD
Methods: In a second experiment, outbred female ICR
mice were assigned to oral and/or epicutaneous antigen
exposure or to placebo groups. Antigen challenge was
administered either by i.p. injection or oral gavage.
Samples were collected and outcomes monitored as in the
first experiment.
0
0
24
34
0.45
OD
C57, naïve (n=3)
Deaths prior to
challenge
Mean temperature change (ºC)
Severe anaphylaxis
(%) upon challenge
Mean Temperature (ºC)
Table 1. Severe adverse
reactions to protocol
0.2
0.1
0
35 (Ch)
0.3
Oral (S), oral (Ch)
Oral (S), IP (Ch)
Oral + EC (S), Oral (Ch)
Oral + EC (S), IP (Ch)
0.15
0
24
34
0
35 (Ch)
0
24
Time after initial sensitization (d)
Time after initial sensitization (d)
34
Time after initial sensitization (d)
Figure 5. Peanut-specific antibody levels in ICR mice. Double sensitization (oral + epicutaneous antigen exposure) led to greater peanut-specific antibody levels than oral exposure alone or placebo. S = sensitization, Ch = challenge, EC =
epicutaneous (via tape stripping), IP = intraperitoneal.
d:
0
7
14
21
28
35
42
Fig. 4. Sensitization and challenge schedule.
Deaths prior to
challenge
16.6
1
50
1
A2: Oral sensitization , IP challenge
(n=6)
B2: Oral + transcutaneous
sensitization, IP challenge (n=6)
40
….. Naïve controls ..…
Oral placebo, IP (Ch)
Oral (S), oral (Ch)
Double placebo, IP (Ch)
Oral + EC (S), oral (Ch)
Oral (S), IP (Ch)
Oral + EC (S), IP (Ch)
38.25
36.5
Double sensitization, IP challenge
34.75
33
Results: Combined oral-plus-epicutaneous sensitization and i.p.
challenge (B2) led to greater mean temperature drop and more severe
anaphylactic reactions (including death) than other protocols.
1
0
10
20
30
Time after challenge dose (min)
Mean temperature change (ºC)
Severe anaphylaxis (%)
upon challenge
Average body temperature in mice
following peanut allergen challenge
Temperature (ºC)
Table 2. Severe adverse reactions
to protocol.
Temperature changes following allergen
challenge: different sensitization and challenge
protocols in ICR mice
0
-1
-2
Double sensitization,
-3
IP challenge
40
-4
A1
A2
B1
B2
C1
C2
D1
D2
Protocol group
Figure 6. Changes in rectal temperature after challenge with complete PE. A) Double sensitization (oral + epicutaneous (EC)) with IP challenge led to rapid, sustained temperature drops. B) Double
sensitization + IP challenge (B2) led to greatest mean temperature drop. Both the route of sensitization and of challenge influenced the outcome (p < 0.0001 and p = 0.0155, respectively). Groups:
Sensitization (S): A = oral, B = oral + epicutaneous, C = oral placebo, D = double placebo; Challenge (Ch): 1 = oral, 2 = IP.
Mean temperature decrease in ICR mice: effect of
vaccination with nanoparticle adjuvants
Change in temperature (ºC)
1.75
0
-1.75
-3.5
-5.25
-7
N
N
N
N
N
N
P3
P3
P2
P2
P1
-e
d
te
y
pt
y
pt
y
pt
na
m
E
m
-P
-e
E
m
-P
-e
E
-P
e
iv
ci
ac
PE
tv
e
P1
na
no
fre
Protocol group
Fig. 7. Application of the selected sensitization protocol to study an
immunotherapy with nanoparticles containing peanut proteins.
Conclusions:
• These results demonstrate for the first time that outbred ICR mice are apt for
the study of peanut hypersensitivity.
• In these mice, epicutaneous antigen exposure intensifies sensitization
(versus oral exposure alone), which, combined with intraperitoneal
challenge, leads to more severe manifestations of peanut-induced
anaphylaxis.
References
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allergy: insights into etiology, prevention, and treatment
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2. Bartnikas LM, Gurish MF, Burton OT, Leisten S, Janssen E,
Oettgen HC, Beaupré J, Lewis CN, Austen KF, Schulte S,
Hornick JL, Geha RS, Oyoshi MK. Epicutaneous sensitization
results in IgE-dependent intestinal mast cell expansion and
food anaphylaxis. J Allergy Clin Immunol. 2013; 131: 451–460
(e1-6).
3. Anagnostou K, Islam I, King Y, Foley L, Pasea L, Bond S,
Palmer C, Deighton J, Ewan P, Clark A. Assessing the efficacy
of oral immunotherapy for the desensitisation of peanut allergy
in children (STOP II): a phase 2 randomised controlled trial.
Lancet. 2014; 383:1297-1304.