Influence of antigen density and TLR ligands on preclinical efficacy of a VLP-based vaccine against peanut allergy.

BACKGROUND: Virus-like particle (VLP) Peanut is a novel immunotherapeutic vaccine candidate for the treatment of peanut allergy. The active pharmaceutical ingredient represents cucumber mosaic VLPs (CuMVTT -VLPs) that are genetically fused with one of the major peanut allergens, Ara h 2 (CuMVTT -Ara h 2). We previously demonstrated the immunogenicity and the protective capacity of VLP Peanut-based immunization in a murine model for peanut allergy. Moreover, a Phase I clinical trial has been initiated using VLP Peanut material manufactured following a GMP-compliant manufacturing process. Key product characterization studies were undertaken here to understand the role and contribution of critical quality attributes that translate as predictive markers of immunogenicity and protective efficacy for clinical vaccine development. METHOD: The role of prokaryotic RNA encapsulated within VLP Peanut on vaccine immunogenicity was assessed by producing a VLP Peanut batch with a reduced RNA content (VLP Peanut low RNA). Immunogenicity and peanut allergen challenge studies were conducted with VLP Peanut low RNA, as well as with VLP Peanut in WT and TLR 7 KO mice. Furthermore, mass spectrometry and SDS-PAGE based methods were used to determine Ara h 2 antigen density on the surface of VLP Peanut particles. This methodology was subsequently applied to investigate the relationship between Ara h 2 antigen density and immunogenicity of VLP Peanut. RESULTS: A TLR 7 dependent formation of Ara h 2 specific high-avidity IgG antibodies, as well as a TLR 7 dependent change in the dominant IgG subclass, was observed following VLP Peanut vaccination, while total allergen-specific IgG remained relatively unaffected. Consistently, a missing TLR 7 signal caused only a weak decrease in allergen tolerability after vaccination. In contrast, a reduced RNA content for VLP Peanut resulted in diminished total Ara h 2 specific IgG responses, followed by a significant impairment in peanut allergen tolerability. The discrepant effect on allergen tolerance caused by an absent TLR 7 signal versus a reduced RNA content is explained by the observation that VLP Peanut-derived RNA not only stimulates TLR 7 but also TLR 3. Additionally, a strong correlation was observed between the number of Ara h 2 antigens displayed on the surface of VLP Peanut particles and the vaccine's immunogenicity and protective capacity. CONCLUSIONS: Our findings demonstrate that prokaryotic RNA encapsulated within VLP Peanut, including antigen density of Ara h 2 on viral particles, are key contributors to the immunogenicity and protective capacity of the vaccine. Thus, antigenicity and RNA content are two critical quality attributes that need to be determined at the stage of manufacturing, providing robust information regarding the immunogenicity and protective capacity of VLP Peanut in the mouse which has translational relevance to the human setting.

Allergen Content of Therapeutic Preparations for Allergen-Specific Immunotherapy of European Paper Wasp Venom Allergy.

Allergy to Polistes dominula (European paper wasp) venom is of particular relevance in Southern Europe, potentially becoming a threat in other regions in the near future, and can be effectively cured by venom immunotherapy (VIT). As allergen content in extracts may vary and have an impact on diagnostic and therapeutic approaches, the aim was to compare five therapeutic preparations for VIT of P. dominula venom allergy available in Spain. Products from five different suppliers were analyzed by SDS-PAGE and LC-MS/MS and compared with a reference venom sample. Three products with P. dominula venom and one product with a venom mixture of American Polistes species showed a comparable band pattern in SDS-PAGE as the reference sample and the bands of the major allergens phospholipase A1 and antigen 5 were assignable. The other product, which consists of a mixture of American Polistes species, exhibited the typical band pattern in one, but not in another sample from a second batch. All annotated P. dominula allergens were detected at comparable levels in LC-MS/MS analysis of products containing P. dominula venom. Due to a lack of genomic information on the American Polistes species, the remaining products were not analyzed by this method. The major Polistes allergens were present in comparable amounts in the majority, but not in all investigated samples of venom preparations for VIT of P. dominula venom allergy.

Venom Immunotherapy: From Proteins to Product to Patient Protection.

In this review, we outline and reflect on the important differences between allergen-specific immunotherapy for inhalant allergies (i.e., aeroallergens) and venom-specific immunotherapy (VIT), with a special focus on Venomil® Bee and Wasp. Venomil® is provided as a freeze-dried extract and a diluent to prepare a solution for injection for the treatment of patients with IgE-mediated allergies to bee and/or wasp venom and for evaluating the degree of sensitivity in a skin test. While the materials that make up the product have not changed, the suppliers of raw materials have changed over the years. Here, we consolidate relevant historical safety and efficacy studies that used products from shared manufacture supply profiles, i.e., products from Bayer or Hollister-Stier. We also consider the characterization and standardization of venom marker allergens, providing insights into manufacturing controls that have produced stable and consistent quality profiles over many years. Quality differences between products and their impacts on treatment outcomes have been a current topic of discussion and further research. Finally, we review the considerations surrounding the choice of depot adjuvant most suitable to augmenting VIT.

Shaping Modern Vaccines: Adjuvant Systems Using MicroCrystalline Tyrosine (MCT®).

The concept of adjuvants or adjuvant systems, used in vaccines, exploit evolutionary relationships associated with how the immune system may initially respond to a foreign antigen or pathogen, thus mimicking natural exposure. This is particularly relevant during the non-specific innate stage of the immune response; as such, the quality of this response may dictate specific adaptive responses and conferred memory/protection to that specific antigen or pathogen. Therefore, adjuvants may optimise this response in the most appropriate way for a specific disease. The most commonly used traditional adjuvants are aluminium salts; however, a biodegradable adjuvant, MCT®, was developed for application in the niche area of allergy immunotherapy (AIT), also in combination with a TLR-4 adjuvant-Monophosphoryl Lipid A (MPL®)-producing the first adjuvant system approach for AIT in the clinic. In the last decade, the use and effectiveness of MCT® across a variety of disease models in the preclinical setting highlight it as a promising platform for adjuvant systems, to help overcome the challenges of modern vaccines. A consequence of bringing together, for the first time, a unified view of MCT® mode-of-action from multiple experiments and adjuvant systems will help facilitate future rational design of vaccines while shaping their success.

The grass pollen season 2014 in Vienna: A pilot study combining phenology, aerobiology and symptom data.

BACKGROUND: Grasses (Poaceae) are one of the largest plant families and are distributed worldwide. Grass pollen allergy is one of the most important pollen allergies affecting large parts of the population worldwide. The grass pollen season itself is special since it is caused by the flowering of various grass species that present unique profiles of allergenicity, which assumingly plays a significant role and impact on grass pollen sensitization profiles for the allergy sufferer. The aim of this study, conducted in Vienna, 2014, was to analyze the possible contribution of prevalent grass species to the grass pollen season and to the symptom load of grass allergy sufferers. METHODS: This was the first study that combines phenological observations (i.e. grass species and their flowering periods) with aerobiological measurements (i.e. daily grass pollen concentrations) in concert with allergic symptoms from local users of the Patient's Hayfever Diary (symptom load index calculation). RESULTS: Both the pollen concentrations and the symptom levels were higher in the first half of the main grass pollen season and therefore show the highest impact on pollen allergy sufferers. Of important note were the following species that are widely distributed in Vienna: Kentucky bluegrass (Poa pratensis), orchard grass (Dactylis glomerata), false oat-grass (Arrhenatherum elatius), fescue grass (Festuca sp.) and perennial rye-grass (Lolium perenne). CONCLUSION: Monitoring different grass species provided evidence for varying contribution in pollination across the main grass pollen season and highlighted the significance this impact may have on pollen allergy sufferers.

A battery of genotoxicity studies with an allergy vaccine adjuvanted with monophosphoryl lipid A (MPL®) for the treatment of grass pollen allergy.

The allergy vaccine Pollinex® Quattro Grass, developed for the prevention/relief of allergic symptoms caused by grass pollen in adults and children, contains extracts of 12 grass pollens and rye cereal (all chemically modified by glutaraldehyde), adsorbed onto L-tyrosine with addition of the immunostimulatory adjuvant monophosphoryl lipid A (MPL®). This paper represents the first publication on genetic toxicology data for such a vaccine. An Ames test using five strains of Salmonella typhimurium at concentrations up to 2000 standardized units (SU) per plate in the absence (-) and presence (+) of metabolic activation (rat liver S9) showed negative results, as did treatment - S9 in the mouse lymphoma assay (MLA). However, a reproducible positive response was noted in the MLA + S9, which could not be attributed to extreme culture conditions, but may have been an artefact of the exogenous metabolizing system. Up to 60% false positive results are reported for the MLA with noncarcinogens. Hence, in vivo genotoxicity studies were conducted. These further assessments comprised a rat bone marrow micronucleus test following subcutaneous (s.c.) doses of 10 000, 20 000 or 40 000 SU kg⁻¹ per day for 2 days, and a comet assay in liver and kidney cells from rats treated with Pollinex® Quattro Grass at 20 000 or 40 000 SU kg⁻¹ per day s.c. for 2 days. Plasma analysis showed evidence of systemic antibodies to Pollinex® Quattro Grass immunogens, but the exposure levels could not be quantified. No evidence of genotoxicity was observed in either of the in vivo studies. Overall, the genotoxicity test results supported safe clinical use of Pollinex® Quattro Grass.

Cross-reactivity in Grasses: Biochemical Attributes Define Exemplar Relevance.

INTRODUCTION: : Broad-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. METHODS: : Grass 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. RESULTS: : Variation 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. DISCUSSION: : None 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.

Reproduction and juvenile animal toxicology studies in the rat with a new allergy vaccine adjuvanted with monophosphoryl lipid A (MPL(®)) for the treatment of grass pollen allergy.

Pollinex(®) Quattro Grass has been developed for the prevention or relief of allergic symptoms caused by pollen in both adults and children. Reproduction and juvenile animal toxicology studies have been performed. Subcutaneous injection on Day 14 prior to pairing and on Days 6 and 13 of gestation to pregnant rats at 2000SU/0.5 mL elicited no signs of maternal or embryo-foetal toxicity. Mating, fertility, fecundity and pup parameters were all unaffected by treatment. Once-weekly subcutaneous administration at ascending doses of 300, 800, 2000 and 2000SU/0.5 mL followed by a 4 week non-dose period to juvenile rats from 3 weeks of age showed no signs of obvious toxicity. As in a previously performed adult animal toxicology study with the vaccine, not unexpected, but relatively minor, immuno-stimulatory effects were seen in this study along with injection site reaction which can largely be attributed to the presence of tyrosine in the formulation.