Inmunogenicidad y capacidad protectora de variantes de la nueva vacuna antialérgica PROLINEM DS / mmunogeni city and protective efficacy of variants of a novel PROLINEM DS anti - allergic vaccine

Introducción: La vacuna antialérgica de segunda generación PROLINEM-DS está compuesta por alérgenos del ácaro Dermatophagoides siboney y la combinación de adyuvantes: proteoliposoma de N. meningitidis B y gel de hidróxido de aluminio. La adsorción del alérgeno es relevante para la seguridad y eficacia clínica de las vacunas adsorbidas en gel de hidróxido de aluminio en estudios previos se demostró la influencia negativa de los iones fosfato en la adsorción del alérgeno. Objetivo: Evaluar la inmunogenicidad y capacidad protectora de cuatro variantes de formulación obtenidas dentro del espacio de diseño de la vacuna PROLINEM DS. Métodos: Se emplearon 4 variantes de formulación con diferentes contenidos de tampón fosfato salino y gel de hidróxido de aluminio. Se administraron a ratones BALB/c 3 dosis subcutáneas una por semana. Luego, los ratones fueron sometidos a reto alergénico por aerosol. Resultados: Todas las variantes indujeron anticuerpos IgG1 e IgG2a alérgeno específico. Este efecto se correlacionó con el balance de citoquinas proinflamatorias Th1/Th2 en los pulmones y en los ganglios. La variante con reducción de tampón fosfato salino y gel de hidróxido de aluminio fue la de mayor índice IgG/IgE después de la vacunación. Esta relación muestra, en una variable, el equilibrio entre los componentes potencialmente bloqueadores y efectores. La tolerancia local en el lugar de la inyección mostró una reducción de los granulomas en los ratones vacunados con menos gel de hidróxido de aluminio. Conclusiones: La reducción del contenido de gel de hidróxido de aluminio y fosfatos se consideran mejoras farmacéuticas sin inconvenientes en cuanto a la inmunogenicidad de esta vacuna con un perfil de seguridad satisfactorio para futuros ensayos clínicos en humanos.

Introduction: The second generation anti allergic vaccine named PROLINEM DS is based on allergens from D. siboney house dust mite and a combination adjuvant containing PL and Alum. Allergen adsorption is relevant both safety and clinical efficacy in alum-adsorbed vaccines. Negative influence of phosphate ions on allergen adsorption was demonstrated in previous researches. Objective: To evaluate immunogenicity and protective efficacy of four variants obtained within design space of PROLINEM DS vaccine. Methods: Four variants were differentiated from each other by both phosphate and alum contents. Balb/c mice were administered with 3 doses by subcutaneous route. Further, mice were subjected to allergen aerosol challenge. Results: Specific IgG1 and IgG2a antibodies were induced by four vaccine variants. It was correlated with pro inflammatory cytokines balance Th1/Th2 both in lungs and lymphatic nodes. Formulation with lower PBS and Alum levels showed the highest IgG/IgE ratio at the end of vaccination schedule. This ratio shows in one variable the balance between potentially blocking and effector components. Mice injected with lower level of Alum showed a reduction of granuloma size in the site of vaccine administration. Conclusion: Decrease both alum and phosphate contents were a pharmaceutical improvement for antiallergic vaccines formulation. Safety and efficacy in this vaccine are crucial for future human clinical trials.

Immunogenicity of a novel anti-allergic vaccine based on house dust mite purified allergens and a combination adjuvant in a murine prophylactic model.

The outer-membrane-derived proteoliposome (PL) of Neisseria meningitidis has been reported as a potent vaccine adjuvant, inducing a Th1-skewed response. This work aimed to assess the immunogenicity of a novel anti-allergic vaccine candidate based on allergens from Dermatophagoides siboney house dust mite and a combination adjuvant containing PL and Alum. In a preventative experimental setting, BALB/c mice were administered with three doses containing 2 µg of Der s1 and 0.4 µg Der s2 allergen, PL and Alum, at 7 days intervals, by subcutaneous route. Furthermore, mice were subjected to an allergen aerosol challenge for 6 consecutive days. Serum IgE, IgG1, and IgG2a allergen-specific antibodies were assessed by ELISA. Cytokine levels in supernatants of D. siboney stimulated lymphocyte cultures and in bronchoalveolar lavage (BAL) were measured by ELISA. Lung tissues were subjected to histological examination. The vaccine prevented the development of both, systemic (IgE) and local allergic responses (featuring lower IL-4, and IL-5 levels in BAL) upon allergen exposure by the inhalant route. Histological examination showed also a diminished allergic inflammatory response in the lungs. After the allergen challenge, cytokine levels in stimulated lymphocyte cultures showed lower values of IL-13 and augmented IFN-γ and IL-10. The vaccine induced a mixed IgG2a/IgG1 antibody response; although only IgG2a was PL-dependent. Both, IgG1/IgE and IgG2a/IgE ratios, showed significantly greater values in vaccinated mice. The findings support a preventative anti-allergic effect associated with the induction of a Th1-like IFN-γ/IL-10 response. IgG1/IgE and IgG2a/IgE ratios could be useful biomarkers for translation into clinical trials.

Aqueous intradermal low-dose house dust mite immunotherapy in tropical settings: a valid cost-effective approach for developing nations?

INTRODUCTION: Aqueous allergen injections, an effective and century-old technique, is considered a second-line approach in daily clinical practice. Inconveniences still surround conventional subcutaneous immunotherapy (SCIT) administration, such as a need for frequent injections, prolonged up-dosing schedules, elevated costs, and the unlikely possibility of a systemic reaction. The intradermal immunotherapy route (IDR) might favorably impact many of the aforementioned issues (Table 1). House dust mite (HDM) allergens are the main perennial sensitizers in the tropics, and as such, are solely employed in immunotherapy treatments. METHODS: We carried out a year-long real-life study in 25 perennial allergic rhinitis children, symptomatic on exposure to house dust, employing an intradermal low-dose allergen mix consisting of 50 ng of Dermatophagoides pteronyssinus/Dermatophagoides farinae and 120 ng of Blomia tropicalis, under a unique cost-wise protocol. Basal symptoms/signs and face Visual Analog Scale (fVAS) scores were recorded for 2 weeks and later compared with those registered throughout the 1-year treatment. Serum-specific IgG4 and IL-10 levels were employed in the assessment of the immune responses. RESULTS: Symptoms/signs and fVAS scores were significantly reduced from days 42 and 49, respectively, and remained so until treatment completion. Increases in specific IgG4's and IL-10 levels reflected significant immune responses. Injections were well tolerated and families reported improved health status (quality of life, QoL). CONCLUSIONS: A unique cost-effective immunotherapy alternative for deprived allergic communities in tropical settings is depicted; further research is needed.

Proteomic identification of allergenic proteins in red oak (Quercus rubra) pollen.

BACKGROUND: Red oak pollen is an important cause of allergic respiratory disease and it is widely distributed in North America and central Europe. To date, however, red oak pollen allergens have not been identified. Here, we describe the allergenic protein profile from red oak pollen. METHODS: Total proteins were extracted from red oak pollen using a modified phenolic extraction method, and, subsequently, proteins were separated by two-dimensional gel electrophoresis (2DE) for both total protein stain (Coomassie Blue) and immunoblotting. A pool of 8 sera from red oak sensitive patients was used to analyze blotted proteins. Protein spots were analyzed by Mass Spectrometry. RESULTS: Electrophoretic pattern of total soluble proteins showed higher intensity bands in the regions of 26-40 and 47-52 kDa. Two dimensional immunoblots using pool sera from patients revealed four allergenic proteins spots with molecular masses in the range from 50 to 55 kDa. Mass spectrometry analysis identified 8 proteins including Enolase 1 and Enolase 1 chloroplastic, Xylose isomerase (X1 isoform), mitochondrial Aldehyde dehydrogenase, UTP-Glusose-1-phosphate uridylyltransferase, Betaxylosidase/alpha-l-arabinofuranosidase and alpha- and beta subunits of ATP synthase. CONCLUSIONS: This study has identified for first time 8 IgE binding proteins from red oak pollen. These findings will pave the way towards the development of new diagnostic and therapeutic modalities for red oak allergy.

In vitro safety of ketotifen as a topical nasal rinse.

BACKGROUND: Ketotifen is a second-generation noncompetitive H1-antihistamine and mast-cell stabilizer. It is commonly used to treat or prevent allergic conjunctivitis, asthma, chronic urticaria, anaphylaxis, mast-cell, and other allergic-type disorders. However, it has never been studied in aspirin-exacerbated respiratory disease (AERD), an aggressive phenotype of chronic rhinosinusitis with nasal polyps, where the mast cell plays a prominent role its pathogenesis. METHODS: Human sinonasal epithelial cells were grown at an air-liquid interface (ALI). Ketotifen powder was dissolved in saline to make 4 test solutions at 1.04, 2.08, 10.4, and 20.8 µg/mL. Control (saline) or ketotifen solution was added apically to ALI cultures from tissue of 5 unique patients, and ciliary beat frequency (CBF) changes were recorded. Lactate dehydrogenase was measured at 24 and 48 hours to estimate long-term cellular toxicity. RESULTS: Apical application of ketotifen at all concentrations was neither ciliotoxic nor ciliostimulatory, with no change in CBF over a period of 15 minutes after application. Cellular toxicity for all concentrations at 24 and 48 hours after application was <3% and <7%, respectively, that of lysed cultures. CONCLUSION: Topical application of ketotifen to an in vitro model of sinonasal epithelium is safe, as evaluated by CBF and lactate dehydrogenase. Ketotifen is neither ciliotoxic nor ciliostimulatory, and no long-term cellular toxicity was observed. Ketotifen may have promise as a topical nasal rinse in the treatment of AERD.