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.

Remote induction of cellular immune response in mice by anti-meningococcal nanocochleates - nanoproteoliposomes.

New adjuvant formulations, based on proteoliposomes <40 nm and cochleates <100 nm, without Al(OH)3 adjuvant, were evaluated regarding their ability to generate Th1 immune response through a Delayed -Type Hypersensitivity Test, at the mouse model, by using a Neisseria meningitidis B protein complex as antigen. The formulations were administered by intramuscular (IM) (2 inoculations - at baseline and after 14 days) and intranasal (IN) (3 inoculations at 7 days) immunization pathways. All IM immunized groups were able to induce similar response to these formulations as well as to VA-MENGOC-BC® vaccine - containing Al(OH)3 adjuvant (used as positive control of the trial). In all groups, the induced inflammation (IP) rate was statistically higher than in the negative control group (CN) (p < 0.05). Immunogenicity, measured by HSR and CD4+ lymphocyte increase was equivalent to the control vaccine and most important, granuloma reactogenicity at the site of injection was eliminated, fact demonstrated by histological study. All groups of animals immunized by IN route showed HSR reactions and statistically significant differences with respect to the CN group. However, IP values were lower, with statistical differences (p < 0.05) for the same adjuvant formulation IM administered, except the AIF2-nCh formulation that generated statistically similar induction (p > 0.05) by both immunization pathways, suggesting it to be the best candidate for the next IN trial. Proteoliposome and cochleate formulations tested were able to mount potent Th-1 immune response, equivalent to the original vaccine formulation, with the advantage of less reactogenicity in the site of the injection, caused by the toxicity of Al(OH)3 adjuvant gel.

Safety of a proteoliposome from Neisseria meningitides as adjuvant for a house dust mite allergy vaccine.

The proteoliposome (PL) of Neisseria meningitidis serogroup B has been reported as a safe and potent vaccine adjuvant, inducing a TH1-skewed response. The present study describes a pre-clinical safety evaluation of an allergy therapeutic vaccine candidate based on purified allergens from Dermatophagoides siboney house dust mite and PL as adjuvant, both components adsorbed onto aluminum hydroxide gel. Two separate studies of acute toxicity evaluation were performed in mice and rabbits, and two repeat-dose studies were conducted in non-sensitized and allergen-sensitized Balb/c mice, respectively. The study in sensitized mice intends to model a therapeutic setting. Aerosolized allergen challenge was used in both settings to model natural respiratory exposure. In the therapeutic setting, mice were administered with three doses containing 2 µg allergen at weekly intervals [subcutaneous route] and subsequently challenged with aerosolized allergen for 6 consecutive days. Parameters of general toxicity effects were assessed via measures of behavior, body weight, food and water consumption, and macroscopic evaluation of organs. Histological examination of organs and the injection site was performed. Potential immunotoxicity effects at the systemic level were assessed by blood eosinophil counting and serum allergen specific IgE by ELISA The vaccine did not produce general or functional toxic effects of significance, at a dose up to 100 µg allergen per kg body weight. An expected local reaction at the injection site was observed, which could be attributed mostly to the immunological effect of aluminum hydroxide. The models implemented here suggest an acceptable safety profile of this vaccine for testing in clinical trials of allergy immunotherapy.

State of the art in developing allergen vaccines in Cuba: prospects of novel adjuvanted vaccines.

Standardized allergen vaccines have been developed and registered as biopharmaceutical products in Cuba. Three different vaccines were obtained from the most relevant allergenic mite species: Dermatophagoides pteronvssinus, Dermatophagoides siboney, and Blomia tropicalis. Immuno-analytical methods based on murine monoclonal antibodies and human IgE antibodies were developed for assessing allergenic potency, composition, and stability. Preclinical and clinical studies showed efficacy and safety in diagnostic prick-tests and subcutaneous immunotherapy in asthmatic patients. New approaches are now undertaken in order to develop new adjuvanted formulations based on liposomes or proteoliposomes from Neisseria meningitidis, and purified allergens; aiming to overcome the drawbacks of conventional immunotherapy.

Modulation of the specific allergic response by mite allergens encapsulated into liposomes.

Liposomes are non toxic and biodegradable lipid vesicles, which are safe and effective adjuvants to induce Th1-skewed immune response. Therefore, the encapsulation of allergens into liposomes could be an attractive alternative for specific allergy immunotherapy. Previously, we obtained DPPC iposomes encapsulating purified allergens from Dermatophagoides siboney, with suitable stability and extremely reduced allergenicity. In this study, Balb/c mice were immunized with allergens ncapsulated into liposomes (LP) and the induced immune response was evaluated in comparison with allergens dissolved in PBS (PBSA) or adsorbed in Alum (AL). The use of Alum or Liposomes induced a strong allergen specific IgG response. However, total IgE serum levels in the AL group were very high, while levels found in LP group were not significantly different from the control group receiving only PBS. The IgG2a/IgG1 subclass ratio was raised in the LP group. Allergen specific IgE, as measured by PCA assay, was similar for LP and PBSA groups, and approximately the half of the reaction size found in AL group. After allergen challenge by inhalation route, peripheral blood and airway eosinophil counts increased significantly in AL, but not in LP group. Additionally, histopathological analysis of lung tissue sections obtained from challenged mice indicated a reduced cellular infiltration in mice immunized with liposomes. These results support the potential use of liposomal formulations for allergen vaccines.