Background: Asthma affects 5.4 million people in the UK. Asthma subgroups are also susceptible to inhalation of fungal spores (Aspergillus fumigatus) and development of pulmonary fungal aspergilloma; presenting a life threatening but poorly understood condition. NHS costs for corticosteroids, bronchodilators and antifungal agents that are only partially effective continue to rise. Allergy immunotherapy development is of great interest as it is specific to the allergen and can harness key adaptive immune T-cells to down-regulate inflammatory responses. Immunotherapy has been used with varying degrees of success for treatment of grass, pollen, venom, cat and dog allergens however to date has not been directed to fungal allergens. The study aims were: 1) to further understand the A. fumigatus allergens and the protein epitopes responsible for generating immune responses. 2) To genotype participating ABPA/SAFS patients to observe any HLA associations. Methods: 37 subjects with fungal sensitivity were recruited to the study which received permission from the local ethics committee (UHSM LREC). Computer bioinformatic predictions using Propred software identified several potential fungal T cell peptide epitopes; of which 8 peptides were soluble and tested in vitro for specific T-cell proliferation responses by flow cytometric analysis. Skin prick tests determined subject responses to fungal allergens including A. fumigatus, and DNA analysis determined subject HLA type. Results: 5 of 8 soluble peptides were Aspergillus fumigatus derived and 3 from Alternaria alternata. All 8 peptides induced higher CD4 proliferative responses in ABPA/SAFS patients, compared to healthy controls from highest significance to lowest as follows: peptide 1.1 > 9.1 > 8.1 > 2.1 > 9.1.1 > 4.1 > 4.1.1 and 10.1.1. 73% subjects elicited skin responses to A. fumigatus. DNA HLA typing identified alleles associated with ABPA/SAFS but not all allele sub types. Discussion: The ABPA/SAFS group consistently raised T-cell responses to fungal peptides compared to controls. This demonstrates peripheral CD4s retain memory for fungal specificity and clearly respond when challenged with fungal epitopes in vitro. This concept underpins the rationale to further characterize the responding CD4 cells and pursuing bioinformatics approaches for immunotherapy investigations for fungal allergy.

Background: Despite the clinical severity and rising prevalence of peanut allergy, there is a marked absence of widespread, practical treatments available for peanut-allergic patients. Peptide immunotherapy, a disease-modifying treatment that uses short peptides recognized by T cells, has been shown to reduce allergic symptoms of allergic rhinoconjunctivitis. This project investigated the ability of peptides from the major peanut allergen Ara h 1 to protect against peanut-induced anaphylaxis and induce immunomodulatory changes in a mouse model. Methods: Mice transgenic for the human leukocyte antigen DRB1*0401 were sensitized to peanut epicutaneously and treated with two intraperitoneal injections of peptides from Ara h 1. Mice were then challenged with intraperitoneal whole peanut and observed for signs of anaphylaxis. Flow cytometry was used to isolate peanut-specific CD4+ T cells labelled with Ara h 1 peptide-loaded tetramers and additional Th1, Th2, and regulatory markers. Results: Peptide-treated mice were protected from severe peanut-induced anaphylaxis. Control mice treated with a sham peptide experienced a mean maximum temperature drop of 3.2°C, while mice treated with Ara h 1 peptides experienced a drop of 1.6°C (p=0.067 vs control). Maximum clinical score was 2.5 in control mice, and 1.4 in treated mice (p=0.0097). Mean hematocrit for control mice was 52.5%, and 47% for treated mice (p=0.013). PD-1+CD4+ T cells were significantly increased in the mesenteric lymph nodes (p = 2.28e-0.05) and spleens (p = 0.014) of peptide-treated mice. MIP1-a+CD4+ T cells were significantly decreased in the peritoneal lavage (p = 0.008). Conclusion: Ara h 1 peptide immunotherapy protected against severe peanut-induced anaphylaxis in a mouse model. Peptide-treated mice experienced significantly reduced drops in core body temperature, clinical signs of allergic reaction, and hemoconcentration. Clinical protection was associated with decreased expression of the pro-inflammatory chemokine macrophage 1-a and increased expression of the surface marker programmed cell death protein 1. / Thesis / Doctor of Philosophy (PhD) / Peanut allergy is a growing public health concern. Its prevalence has doubled in the past 10 years and currently stands at 2%. Reactions to peanut account for the majority of food-induced fatal allergic reactions, termed anaphylaxis. Currently, there are no treatments available for patients with peanut allergy. Healthcare workers can only offer peanut-allergic patients advice on peanut avoidance and rescue medications in case of accidental ingestion. This research project investigated the ability of a new treatment called peptide immunotherapy to prevent severe allergic reactions to peanut in a mouse model of peanut allergy. Peptide treatment uses small portions of the peanut allergen to shift the immune response from pro-inflammatory to anti-inflammatory. After peptide treatment, peanut-allergic mice were protected from severe allergic reactions in response to peanut and their immune cells produced lower levels of pro- inflammatory molecules.

Allergen-reactive CD4+ T cells are implicated in the pathogenesis of allergic disease. Peptide immunotherapy (PIT) involves therapeutic administration of short immunodominant peptides from within the protein allergen to which CD4+ T cell responses are directed. This approach can induce tolerance of allergen-reactive CD4+ T cells, while negating the risk of severe allergic reactions associated with whole allergen specific immunotherapy. PIT therefore holds promise as a diseasemodifying treatment for allergic patients. However, further information regarding the mechanisms of action of PIT are required to aid translation to the allergy clinic. Chicken ovalbumin (OVA) is a commonly used model allergen in mouse models of allergic airways inflammation (AAI). Trackable, T cell receptor transgenic T cells recognizing the immunodominant 323-339 peptide of OVA (pOVA) allow mechanistic investigation of PIT in response to pOVA. This thesis investigated the hypothesis that strong, systemic T cell responses induced by intravenous administration of soluble pOVA will induce i) tolerance to pOVA and ii) linked suppression to any additional OVA T cell epitopes, hence improving OVA-induced AAI. Contrary to the hypothesis, intravenous pOVA PIT did not improve disease in a C57BL/6 model of OVA-induced AAI. Models of OVA-induced allergic sensitisation and AAI were therefore developed incorporating trackable CD4+ pOVA-reactive T cells (OT-II cells). pOVA PIT induced tolerance of these cells in an allergic sensitisation setting, but had limited impact on the overall OVA response. Yet, in a model of AAI driven solely by Th2 polarised CD4+ OT-II cells, pOVA PIT did improve disease. It was concluded that, in non-transgenic C57BL/6 mice, CD4+ T cells responding to additional epitope(s) within OVA were important in driving disease and that these T cells were not subject to linked suppression following pOVA PIT. Using a panel of overlapping peptides constituting the sequence of OVA, a novel CD4+ epitope within OVA was characterised. The effects of PIT using pOVA in combination with a peptide containing this additional epitope on OVA-induced AAI were then assessed. Findings from this project therefore hold importance for future mechanistic work surrounding PIT in allergic disease.

Asthma is defined as reversible airflow obstruction and an estimated 1-in-3 Canadians will be diagnosed over their lifetime. Many clinical phenotypes of asthma exist, but allergic asthma is the most common presentation. Despite effective therapies, approximately 65% of Canadian asthmatics have poorly controlled disease. Thus, there remains pressing need to develop disease modifying therapies. Allergen-specific immunotherapy (SIT) is a disease-modifying therapy for allergic disease that consists of repeatedly administering doses of allergen, to an allergic individual; over 100 years of clinical use, SIT has been demonstrated to reduce symptoms of disease both during and after cessation of therapy. Widespread clinical uptake of SIT has been limited by the risk of developing anaphylaxis as a response to therapy. Peptide immunotherapy is a derivation of SIT, that attempts to retain the disease-modifying benefits, while lessening the risk of anaphylaxis, by treating subjects with allergen-derived T-cell peptide epitopes. Peptide immunotherapy has been demonstrated to reduce symptoms of allergic disease in treated subjects; however, it remains unknown how administration of a single (or several) T-cell epitopes can modulate immune responses to entire complex allergens. Additionally, how genetic diversity in peptide epitope presentation effects the development of immune tolerance is unknown. In this thesis, we sought to characterize these mechanisms of peptide immunotherapy; the hypothesis was, “The induction of immunosuppression by peptide immunotherapy involves the infectious spread of tolerance beyond the treatment epitope, and is dependent upon treatment peptide dose and affinity to MHC”. Through the definition of these mechanistic traits we hoped to expedite and inform the design of future peptide based therapeutics. The studies presented within this thesis examine the topic of immune modulation of allergic disease in mouse models, and have focused upon broadly pertinent characteristics of immune modulation, such as the number, dose and affinity of immunomodulatory epitopes. / Thesis / Doctor of Philosophy (PhD) / Asthma is a disease of the airways that can cause difficulties in breathing. In some people, asthma develops because their immune system reacts in an uncontrolled manner to common environmental proteins, called allergens. Whole allergen immunotherapy is a treatment for asthma, where asthmatic people are injected with doses of allergen until their immune system no longer responds to (or ‘tolerates’) the allergen. In some people, injection of allergen can lead to a life-threatening immune response known as ‘anaphylaxis’. Peptide-immunotherapy is a form of whole allergen immunotherapy where people are given small fragments of the allergen (a ‘peptide’) rather than the whole allergen. The benefit of peptide immunotherapy is that the treatment peptides are too small to cause anaphylaxis, but remain large enough to teach the immune system. In this thesis, we examined how treatment with small peptides teaches the immune system to tolerate the larger and more complex whole allergen.