Peanut T Cell Epitope Discovery: Ara h1 and Ara h3
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804 Peanut T Cell Epitope Discovery: Ara h1 and Ara h3 Dr. Manish Ramesh, MD, PhD 1 , Madhan Masilamani, PhD 2 , Dr. George N. Konstantinou, MD, PhD, MSc 3,4 , Jay A. Lieberman, MD 5 , Hugh A. Sampson, MD, FAAAAI 6 , Dr. Madhundra Sivakumar 1 , Dr. Araya Yuenyongviwat, MD 7 , Dr. Mariona Pascal, PhD 8 ; 1 Mount Sinai School ofMedicine, New York, NY, 2 Icahn School of Medicine at Mount Sinai, New York, NY, 3 424 General Military Training Hospital, Thessalo- niki, Greece, 4 First Pediatric Department, Aristotle University of Thessa- loniki, Hippokration General Hospital, Thessaloniki, Greece, 5 University of Tennessee, Memphis, TN, 6 Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY, 7 Prince of Songkla University, Songkhla, Thailand, 8 Servei d’Immunologia, Centre de Diagnostic Biomedic. Hospi- tal Clinic., Barcelona, Spain. RATIONALE: Peptide-based immunotherapy is a viable modality that circumvents IgE-mediated adverse reactions. Here we identify candidate peptides of Ara h1 and 3 that display promiscuous binding to MHC class II and induce Th2 cytokine production by T cells. METHODS: A two-step peptide screening process was employed. In sil- ico MHC class II binding prediction was performed using NetMHCIIpan 2.0 (peptide length 15, 1-mer offset) using the most abundant class II al- leles in our population. Proimmune RevealÒ (in vitro MHC class II peptide reporter) assay was performed using synthesized 15-mer peptides offset by 5-mer spanning the protein. One hundred patients with an unequivocal his- tory of peanut allergy or IgE level >95% predictive of peanut allergy were enrolled, PBMCs collected, and 3 H-Thymidine T-cell stimulation assay us- ing pre-selected peptides were performed. IL4, IL13, IL5, IFNg and TNFa were measured in culture supernatants using the luminex platform. RESULTS: Comparison of in vitro and in silico results showed good concordance for both Ara h1 and Ara h3. A pool of 36 Ara h1 peptides was selected for T-cell stimulation assays and confirmed these results. In silico analysis predicted promiscuous binding to different HLA types by several peptides. This was also seen in ex vivo T-cell stimulation assays. In combination with cytokines secreted in T-cell assays, we have identified 4 vaccine candidate Ara h1 peptides. Using a similar approach, we have also identified 4 Ara h3 vaccine candidate peptides. CONCLUSIONS: Pre-selection of peptides in combination with conven- tional methods was an effective strategy for identifying peanut T-cell vaccine candidates. 805 Microparticles Encapsulated With Antigen Protect Against Sensitization and Reduce Anaphylactic Reactivity In a Food Allergy Model Dr. Karen B. Chien, PhD 1 , Dr. Paul Bryce, PhD 2 ; 1 Northwestern Univer- sity, Chicago, IL, 2 Division of Allergy-Immunology, Department of Med- icine, Feinberg School of Medicine, Northwestern University, Chicago, IL. RATIONALE: Treatment of food allergy through oral immunotherapy has shown promise, but patients exhibit a high frequency of adverse reactions. Desensitization, rather than tolerance, also seems the likely mechanism. We previously demonstrated that chemical-coupling of anti- gen to splenocytes promoted tolerance and inhibited experimental food allergy; however, this method has limitations for therapy. Therefore, we developed biodegradable microparticles as antigen carriers and examined their use in allergic models. METHODS: Poly(lactic-co-glycolic acid) (PLGA) microparticles encap- sulated with ovalbumin (OVA) were fabricated using double emulsion techniques and characterized for antigen release. Their effects on immune sensitization were determined by prophylactic treatment prior to intraper- itoneal OVA/alum, while their ability to inhibit food allergy responses was assessed using a cholera toxin-driven model with treatment after estab- lished reactivity. RESULTS: PLGA microparticles encapsulating OVAwere homogeneous in size (1-20 microns) and exhibited a sustained release of antigen over several weeks. Pretreatment of animals with different microparticle doses prevented increases in OVA-specific IgE and Th2-associated cytokine responses. Administration to fully sensitized mice, established by anaphylaxis upon challenge, triggered no immediate reactions and prevented anaphylactic symptoms and body temperature changes on challenge after only 1 week. Further studies are ongoing to investigate the mechanisms and efficacy of this therapeutic approach to anaphylaxis. CONCLUSIONS: We have fabricated biodegradable microparticle carriers as an antigen delivery system. Our data suggests that these microparticles are capable of preventing sensitization and can be administered into sensitized animals safely. Since these microparticles ablate subsequent anaphylactic reactions to antigen exposure, they may be a novel approach to treating food allergy. 806 Over-The-Counter Dietary Supplements Genistein and Ipriflavone Suppress Peanut Allergy Symptoms Ms. Lisa Chang, BS 1 , Ms. Mohanapriya Kamalakannan, MS 1 , Mr. Matthew Stadler 1 , Hugh A. Sampson, MD, FAAAAI 2 , Madhan Masilamani, PhD 1 ; 1 Icahn School of Medicine at Mount Sinai, New York, NY, 2 Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY. RATIONALE: Genistein and Ipriflavone are popular dietary supplements used for the treatment of post-menopausal disorders. Both genistein and ipriflavone belong to a group of anti-inflammatory compounds called isoflavonoids. Ipriflavone is an anabolic synthetic daidzein-derivative. We tested the effect of these molecules on peanut allergic mouse models and in ex vivo basophil degranulation in food allergic subjects. METHODS: 6-8 week old female C3H/HeJ mice were fed a soy-free diet (n528), diet containing 1500 ppm each of genistein (n514), ipriflavone (n517) or genistein+ipriflavone (n511). Mice were sensitized, boosted and challenged orally with peanut extract. The allergic symptoms (anaphylaxis scores and body temperature) were recorded after 60 min of challenge at week 10. Whole blood from 6 peanut- and 6 milk allergic subjects was pre-incubated with 100uM genistein, ipriflavone or genistei- n+ipriflavone for 30 min. Basophil degranulation assays were performed in the presence of allergenic extracts+/-isoflavones. RESULTS: Dietary genistein+ipriflavone significantly suppressed pea- nut-induced anaphylaxis in mice compared to soy-free diet (mean scores: 0.18 vs 1.13, p<0.001; mean temperature change: 0.6 vs -1.6, p<0.001). Switching from soy-free diet to genistein+Ipriflavone diet in ‘‘peanut- allergic’’ mice reduced the allergic symptoms. Ipriflavone+genistein dose- dependently suppressed anti-IgE mediated basophil degranulation and suppressed allergen-induced degranulation in food allergic subjects (mean fold change compared to control stimulation: 11 vs 18 for peanut, p<0.01 and 6 vs 11 for milk, p<0.001). CONCLUSIONS: Unlike soy-derived isoflavone mixtures, synthetic genistein and ipriflavone are well documented to be safe for humans and allergen-free. We are currently investigating whether dietary Ipriflavone and genistein supplementation can be used therapeutically for food allergy. J ALLERGY CLIN IMMUNOL VOLUME 133, NUMBER 2 Abstracts AB233 MONDAY
Transcript of Peanut T Cell Epitope Discovery: Ara h1 and Ara h3
J ALLERGY CLIN IMMUNOL
VOLUME 133, NUMBER 2
Abstracts AB233
MONDAY
804 Peanut T Cell Epitope Discovery: Ara h1 and Ara h3Dr. Manish Ramesh, MD, PhD1, Madhan Masilamani, PhD2,
Dr. George N. Konstantinou, MD, PhD, MSc3,4, Jay A. Lieberman, MD5,
Hugh A. Sampson, MD, FAAAAI6, Dr. Madhundra Sivakumar1,
Dr. Araya Yuenyongviwat, MD7, Dr. Mariona Pascal, PhD8; 1Mount Sinai
School of Medicine, New York, NY, 2Icahn School of Medicine at Mount
Sinai, New York, NY, 3424 General Military Training Hospital, Thessalo-
niki, Greece, 4First Pediatric Department, Aristotle University of Thessa-
loniki, Hippokration General Hospital, Thessaloniki, Greece, 5University
of Tennessee, Memphis, TN, 6Pediatrics, Icahn School of Medicine at
Mount Sinai, New York, NY, 7Prince of Songkla University, Songkhla,
Thailand, 8Servei d’Immunologia, Centre de Diagnostic Biomedic. Hospi-
tal Clinic., Barcelona, Spain.
RATIONALE: Peptide-based immunotherapy is a viable modality that
circumvents IgE-mediated adverse reactions. Here we identify candidate
peptides of Ara h1 and 3 that display promiscuous binding to MHC class II
and induce Th2 cytokine production by T cells.
METHODS: A two-step peptide screening process was employed. In sil-
ico MHC class II binding prediction was performed using NetMHCIIpan
2.0 (peptide length 15, 1-mer offset) using the most abundant class II al-
leles in our population. ProimmuneReveal� (in vitroMHCclass II peptide
reporter) assay was performed using synthesized 15-mer peptides offset by
5-mer spanning the protein. One hundred patients with an unequivocal his-
tory of peanut allergy or IgE level >95% predictive of peanut allergy were
enrolled, PBMCs collected, and 3H-Thymidine T-cell stimulation assay us-
ing pre-selected peptides were performed. IL4, IL13, IL5, IFNg and TNFa
were measured in culture supernatants using the luminex platform.
RESULTS: Comparison of in vitro and in silico results showed good
concordance for both Ara h1 and Ara h3. A pool of 36 Ara h1 peptides
was selected for T-cell stimulation assays and confirmed these results. In
silico analysis predicted promiscuous binding to different HLA types by
several peptides. This was also seen in ex vivo T-cell stimulation assays.
In combination with cytokines secreted in T-cell assays, we have identified
4 vaccine candidate Ara h1 peptides. Using a similar approach, we have
also identified 4 Ara h3 vaccine candidate peptides.
CONCLUSIONS: Pre-selection of peptides in combination with conven-
tional methods was an effective strategy for identifying peanut T-cell
vaccine candidates.
805 Microparticles Encapsulated With Antigen Protect AgainstSensitization and Reduce Anaphylactic Reactivity In a FoodAllergy Model
Dr. Karen B. Chien, PhD1, Dr. Paul Bryce, PhD2; 1Northwestern Univer-
sity, Chicago, IL, 2Division of Allergy-Immunology, Department of Med-
icine, Feinberg School of Medicine, Northwestern University, Chicago,
IL.
RATIONALE: Treatment of food allergy through oral immunotherapy
has shown promise, but patients exhibit a high frequency of adverse
reactions. Desensitization, rather than tolerance, also seems the likely
mechanism. We previously demonstrated that chemical-coupling of anti-
gen to splenocytes promoted tolerance and inhibited experimental food
allergy; however, this method has limitations for therapy. Therefore, we
developed biodegradable microparticles as antigen carriers and examined
their use in allergic models.
METHODS: Poly(lactic-co-glycolic acid) (PLGA) microparticles encap-
sulated with ovalbumin (OVA) were fabricated using double emulsion
techniques and characterized for antigen release. Their effects on immune
sensitization were determined by prophylactic treatment prior to intraper-
itoneal OVA/alum, while their ability to inhibit food allergy responses was
assessed using a cholera toxin-driven model with treatment after estab-
lished reactivity.
RESULTS: PLGA microparticles encapsulating OVAwere homogeneous
in size (1-20 microns) and exhibited a sustained release of antigen over
several weeks. Pretreatment of animals with different microparticle doses
prevented increases in OVA-specific IgE and Th2-associated cytokine
responses. Administration to fully sensitized mice, established by
anaphylaxis upon challenge, triggered no immediate reactions and
prevented anaphylactic symptoms and body temperature changes on
challenge after only 1 week. Further studies are ongoing to investigate
the mechanisms and efficacy of this therapeutic approach to anaphylaxis.
CONCLUSIONS: We have fabricated biodegradable microparticle
carriers as an antigen delivery system. Our data suggests that these
microparticles are capable of preventing sensitization and can be
administered into sensitized animals safely. Since these microparticles
ablate subsequent anaphylactic reactions to antigen exposure, they may be
a novel approach to treating food allergy.
806 Over-The-Counter Dietary Supplements Genistein andIpriflavone Suppress Peanut Allergy Symptoms
Ms. Lisa Chang, BS1, Ms. Mohanapriya Kamalakannan, MS1,
Mr. Matthew Stadler1, Hugh A. Sampson, MD, FAAAAI2, Madhan
Masilamani, PhD1; 1Icahn School of Medicine at Mount Sinai, New
York, NY, 2Pediatrics, Icahn School of Medicine at Mount Sinai, New
York, NY.
RATIONALE: Genistein and Ipriflavone are popular dietary supplements
used for the treatment of post-menopausal disorders. Both genistein and
ipriflavone belong to a group of anti-inflammatory compounds called
isoflavonoids. Ipriflavone is an anabolic synthetic daidzein-derivative. We
tested the effect of these molecules on peanut allergic mousemodels and in
ex vivo basophil degranulation in food allergic subjects.
METHODS: 6-8 week old female C3H/HeJ mice were fed a soy-free diet
(n528), diet containing 1500 ppm each of genistein (n514), ipriflavone
(n517) or genistein+ipriflavone (n511). Mice were sensitized, boosted
and challenged orally with peanut extract. The allergic symptoms
(anaphylaxis scores and body temperature) were recorded after 60 min
of challenge at week 10. Whole blood from 6 peanut- and 6 milk allergic
subjects was pre-incubated with 100uM genistein, ipriflavone or genistei-
n+ipriflavone for 30min. Basophil degranulation assays were performed in
the presence of allergenic extracts+/-isoflavones.
RESULTS: Dietary genistein+ipriflavone significantly suppressed pea-
nut-induced anaphylaxis in mice compared to soy-free diet (mean scores:
0.18 vs 1.13, p<0.001; mean temperature change: 0.6 vs -1.6, p<0.001).
Switching from soy-free diet to genistein+Ipriflavone diet in ‘‘peanut-
allergic’’ mice reduced the allergic symptoms. Ipriflavone+genistein dose-
dependently suppressed anti-IgE mediated basophil degranulation and
suppressed allergen-induced degranulation in food allergic subjects (mean
fold change compared to control stimulation: 11 vs 18 for peanut, p<0.01
and 6 vs 11 for milk, p<0.001).CONCLUSIONS: Unlike soy-derived isoflavone mixtures, synthetic
genistein and ipriflavone are well documented to be safe for humans and
allergen-free. We are currently investigating whether dietary Ipriflavone
and genistein supplementation can be used therapeutically for food allergy.
