IntroductionBroad-spectrum grass pollen immunotherapies contain large numbers of allergenic proteins from multiple species. The principle of homologous grouping is used as a tool to assist in the standardization of allergen immunotherapy. This study reviews the principle of homologous grouping, questions what an exemplar grass should be, and queries whether a 1-way system of inferring homology is appropriate.MethodsGrass pollens were extracted and analyzed using a variety of techniques, including enzyme-linked immunosorbent assay, Bradford protein assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and quantitative analysis of Western blots.ResultsVariation in protein content, IgG, IgE, and Phl p 5 reactivity is evident among all grasses analyzed. There is significant evidence of similarity but also disparity consistent with variation resulting from evolutionary change. Proprietary software called Gel Electrophoresis Protein Profile Analysis has been developed, which highlights that each grass exhibits a greater than 55% similarity measure; this is considered high similarity.DiscussionNone of the grass species examined display an identical biological profile. However, data indicate that there is a high degree of homology, and Crested Dogstail is similar to each of the other 12 species analyzed; these levels of similarity can only be possible because of molecular profile and extensive sharing of epitopes. These data are considered to be sufficient to include Crested Dogstail within the sweet grasses group of the Pooideae family; however, the subtle differences in grasses also justify the inclusion of multiple species to create a broad-spectrum immunotherapy.

IntroductionIntratumoral injections of novel therapeutics can activate tumor antigen-specific T cells for locoregional tumor control and may even induce durable systemic protection (against distant metastases) via recirculating T cells. Here we explored the possibility of a universal immunotherapy that promotes T-cell responses in situ and beyond, upon intratumoral injection of nanoparticles formulated with micron-sized crystals.MethodsCucumber mosaic virus-like particles containing a tetanus toxin peptide (CuMVTT) were formulated with microcrystalline tyrosine (MCT) adjuvant and injected directly in B16F10 melanoma tumors. To further enhance immunogenicity, we loaded the nanoparticles with a TLR7/8 ligand and incorporated a universal tetanus toxin T-helper cell peptide. We assessed therapeutic efficacy and induction of local and systemic immune responses, including RNA sequencing, providing broad insight into the tumor microenvironment and correlates of protection.ResultsMCT crystals were successfully decorated with CuMVTT nanoparticles. This ‘immune-enhancer’ formed immunogenic depots in injected tumors, enhanced polyfunctional CD8+ and CD4+ T cells, and inhibited B16F10 tumor growth locally and systemically. Local inflammation and immune responses were associated with upregulation of genes involved in complement activation and collagen formation.ConclusionsOur new immune-enhancer turned immunologically cold tumors into hot ones and inhibited local and distant tumor growth. This type of immunotherapy does not require the identification of (patient–individual) relevant tumor antigens. It is well tolerated, non-infectious, and affordable, and can readily be upscaled for future clinical testing and broad application in melanoma and likely other solid tumors.

BackgroundGrass allergy immunotherapies often consist of a mix of different grass extracts, each containing several proteins of different physiochemical properties; however, the subtle contributions of each protein are difficult to elucidate. This study aimed to identify and characterize the group 1 and 5 allergens in a 13 grass extract and to standardize the extraction method.MethodsThe grass pollens were extracted in isolation and pooled and also in combination and analyzed using a variety of techniques including enzyme-linked immunosorbent assay, liquid chromatog-raphy-mass spectrometry, and sodium dodecyl sulfate-polyacrylam-ide gel electrophoresis.ResultsGold-staining and IgE immunoblotting revealed a high degree of homology of protein bands between the 13 species and the presence of a densely stained doublet at 25–35 kD along with protein bands at approximately 12.5, 17, and 50 kD. The doublet from each grass species demonstrated a high level of group 1 and 5 interspecies homology. However, there were a number of bands unique to specific grasses consistent with evolutionary change and indicative that a grass mix immunotherapeutic could be considered broad spectrum.ConclusionsSodium dodecyl sulfate-polyacrylamide gel electro-phoresis and IgE immunoblotting showed all 13 grasses share a high degree of homology, particularly in terms of group 1 and 5 allergens. IgE and IgG enzyme-linked immunosorbent assay potencies were shown to be independent of extraction method.

BackgroundGrass allergy immunotherapies often consist of a mix of different grass extracts each containing several proteins of different physiochemical properties; however the subtle contributions of each protein are difficult to elucidate. This study aimed to identify and characterise the group 1 and 5 allergens in a 13 grass extract and to standardise the extraction method.MethodsThe grass pollens were extracted in isolation and pooled and also in combination and analysed using a variety of techniques including enzyme-linked immunosorbent assay (ELISA), liquid chromatography-mass spectrometry (LC-MS) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).ResultsGold-staining and IgE immunoblotting revealed a high degree of homology of protein bands between the 13 species and the presence of a densely stained doublet at 25 to 35 kD along with protein bands at approximately 12.5, 17 and 50 kD. The doublet from each grass species demonstrated a high level of group 1 and 5 interspecies homology. However, there were a number of bands unique to specific grasses consistent with evolutionary change and indicative that a grass mix immunotherapeutic could be considered broad spectrum.ConclusionsSDS-PAGE and IgE immunoblotting showed all 13 grasses share a high degree of homology particularly in terms of group 1 and 5 allergens. IgE and IgG ELISA potencies were shown to be independent of extraction method.

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