Understanding the triggers of pollen allergy in Tasmania : a novel molecular approach

Background
Pollen allergy is a global health concern. It contributes to a significant economic burden, can cause prolonged suffering of individuals and in some situations, mortality. Pollen allergy, in this case allergic rhinitis and asthma, management can be facilitated through pre-emptive medication, avoidance of triggers and long-term immunotherapy. These strategies require two specific categories of knowledge i) which pollen are you allergic to and ii) when is that allergen present in the air.
Tasmania, a state with high rates and burden of pollen allergy, demonstrates three notable gaps limiting the acquisition of these two forms of knowledge. The first gap relates to pollen allergy diagnosis. Clinicians are not currently able to diagnose allergy to pollen taxa that do not currently have a diagnostic test. In Hobart, Jones et al. (2020) identified an association between the incidence of smartphone-reported allergic rhinitis symptoms and atmospheric concentration of one such taxon, Dodonaea. There is no current diagnostic test for Dodonaea allergy and therefore there is no understanding of the molecular basis of its potential allergenicity or its potential public health burden. The remaining gaps relate to airborne pollen monitoring. First, current pollen monitoring practice, in some situations, does not provide sufficient taxonomic resolution to distinguish allergenic groups of some pollen taxa. Finally, with burgeoning evidence of a large degree of variation in pollen allergen content (pollen potency), atmospheric pollen concentrations, as delivered by pollen monitoring, may not directly correlate with the trigger of allergy, allergen. In this thesis, I used a variety of methods, including a scoping review as well as two novel molecular biology techniques to address these three gaps.

Specific Objectives

1. Expanding the taxonomic scope of pollen allergy diagnostic tools:
   - This will be addressed with a novel immunoassay (Chapter 2).
2. Improving pollen monitoring precision in terms of taxonomic resolution of taxa in the air and their degree of allergenicity:
   - This will be addressed with a novel metabarcoding pipeline (Chapters 3 and 4) and a scoping review (Chapter 5).

Chapter 2
I investigated a novel method, the halogen immunoassay (HIA), that might be able to determine the allergenicity of potentially novel aeroallergens, such as Dodonaea, without requiring a commercial allergen extract. I optimised this method to detect the binding of participant IgE, a proxy for allergic sensitisation, to rye grass (Lolium perenne) pollen grains. After this, I applied HIAs to a cohort of hay fever sufferers in Hobart, using a battery of known allergenic plants in addition to Dodonaea. I was able to demonstrate positive HIA results to rye grass but not to other taxa, including Dodonaea. When comparing the results to other diagnostics that utilise allergen extracts, HIAs detected a similar cohort with positive results with approximately half the sensitivity to specific IgE tests and skin prick tests. Comparing the results of the HIA method using allergen extracts rather than pollen grains suggested that singular pollen grains might explain the lower sensitivity of the pollen HIA rather than the method itself. Furthermore, I demonstrated positive HIA results using extracts from Betula, Acacia and Allocasuarina. Therefore, with further optimisation, I hypothesise that the HIA may be able to demonstrate a positive result for single pollen grains to other taxa, and if it is allergenic, Dodonaea.
Chapters 3 and 4
I optimised (Chapter 3) and implemented (Chapter 4) a novel metabarcoding pipeline to characterise genus-level patterns of key allergenic families, Poaceae (grasses), Cupressaceae (cypresses) and Myrtaceae (includes Eucalyptus) in the air of Hobart. I took two years of weekly air samples, using the ITS2 and trnL-trnF metabarcodes, alongside routine morphological pollen counts. I found that the peak grass pollen season of November-January was dominated by temperate grass pollen genera such as Poa, Bromus and Holcus. Subtropical grasses such as Cynodon only feature minimally off-season. The bulk of Cupressaceae pollen was from introduced cypresses such as Cupressus and Juniperus, with only small amounts of native Callitris being detected. The burden of Myrtaceae pollen was found to be Eucalyptus with Melaleuca and other genera being less detected. Additionally, I found trnL-trnF to be a useful accessory metabarcode to the more accepted and utilised marker ITS2. In this study, I provided key information on aeroallergens present in the air, as opposed to broad taxonomic groups and I further highlighted the broad utility of metabarcoding studies.
Chapter 5
Through a scoping review, I confirmed that pollen potency is a highly variable phenomenon. I found a large degree of taxonomic and geospatial variation. There was also extensive inter- and intra-seasonal/temporal variation. I also found no consistent signals about the factors that might affect or drive potency variation, meaning it is currently impossible to model and predict. Finally, I confirmed the minor impact that variation in pollen potency may have on pollen monitoring practice. This was demonstrated through the slightly stronger relationship of disease metrics with atmospheric allergen concentration than with atmospheric pollen concentration. Overall, this chapter highlighted that there is a great deal of research gaps required to better understand this emerging field.
Conclusions
The findings of my thesis have broad implications for both future research and the management of pollen allergy symptoms, especially those in Tasmania. By disentangling the seasonal patterns of aerial pollen of allergenic subfamilies of grass, individuals with temperate and subtropical grass allergies will be able to identify the broad seasonal patterns and daily risk levels and take measures to avoid symptoms. I did not successfully adapt the HIA method to address the allergenicity of potentially novel aeroallergens, such as Dodonaea. However, I did highlight the ways HIAs can be optimised to meet this need and I hypothesised that it may still meet this goal. I generated a comprehensive and well-cited synthesis of the extent and implications of variable pollen potency. Throughout this thesis, I have highlighted the benefits of using molecular approaches to address the management of the multifaceted and burdensome disease of pollen allergy, especially in a Tasmanian context.