Block Copolymer/DNA Vaccination Induces a Strong Allergen-Specific Local Response in a Mouse Model of House Dust Mite Asthma

<div><p>Background</p><p>Allergic asthma is caused by abnormal immunoreactivity against allergens such as house dust mites among which <i>Dermatophagoides farinae</i> (Der f) is a common species. Currently, immunotherapy is based on allergen administration, which has variable effect from patient to patient and may cause serious side effects, principally the sustained risk of anaphylaxis. DNA vaccination is a promising approach by triggering a specific immune response with reduced allergenicity.</p><p>Objective</p><p>The aim of the study is to evaluate the effects of DNA immunization with Der f1 allergen specific DNA on allergic sensitization, inflammation and respiratory function in mice.</p><p>Methods</p><p>Mice were vaccinated 28 and 7 days before allergen exposure with a Der f1-encoding plasmid formulated with a block copolymer. Asthma was induced by skin sensitization followed by intra-nasal challenges with Der f extract. Total lung, broncho-alveolar lavage (BAL) and spleen cells were analyzed by flow cytometry for their surface antigen and cytokine expression. Splenocytes and lung cell IFN-γ production by CD8+ cells in response to Der f CMH1-restricted peptides was assessed by ELISPOT. IgE, IgG1 and IgG2a were measured in serum by ELISA. Specific bronchial hyperresponsiveness was assessed by direct resistance measurements.</p><p>Results</p><p>Compared to animals vaccinated with an irrelevant plasmid, pVAX-Der f1 vaccination induced an increase of B cells in BAL, and an elevation of IL-10 and IFN-γ but also of IL-4, IL-13 and IL-17 producing CD4+ lymphocytes in lungs and of IL-4 and IL-5 in spleen. In response to CD8-restricted peptides an increase of IFN-γ was observed among lung cells. IgG2a levels non-specifically increased following block copolymer/DNA vaccination although IgE, IgG1 levels and airways resistances were not impacted.</p><p>Conclusions & Clinical Relevance</p><p>DNA vaccination using a plasmid coding for Der f1 formulated with the block copolymer 704 induces a specific immune response in the model of asthma used herein.</p></div

Immunization protocols against Der f1 in asthmatic mice.

<p>pVAX-Der f1 or pCMV-βgal plasmid were injected i.m at days −28 and −7. Mice were then epicutaneously sensitized and intranasally challenged with total extract of HDM. Analyses were performed on day 35.</p

Der f1 and βgal expression assessment.

<p>Five days after the last vaccine injection, Der f1 and βgal gene expression were verified by RT-PCR on skeletal muscle RNA using specific primers (n = 3 per group). This experience was done for both DNA vaccination conditions (10 µg and 5 µg of plasmid preparation).</p

Effect of immunization protocol on BAL inflammation of non asthmatic non vaccinated mice (light grey bar, n = 8), Der f mice (open bar, n = 9), pCMV-βgal (grey bar, n = 7) and pVAX-Der f1 mice (black bar, n = 7) when using 10 µg of DNA.

<p>The total number of cells was determined by cell count on Kova slides. The cellular composition was established by flow cytometry. Results are expressed as absolute number of cells, as the mean and standard deviation for each group. **p<0,01 and *** p<0,001 by Mann Whitney test.</p

Flow cytometry mix for detection of cytokine produced by lung T cells.

<p>Flow cytometry mix for detection of cytokine produced by lung T cells.</p

Effect of immunization protocol on T cells cytokines secretion of Der f mice (open bar, n = 6), pCMV-βgal (grey bar, n = 7) and pVAX-Der f1 (black bar, n = 7) when using 10 µg of DNA.

<p>One day after the last airway allergen challenge, mice were sacrificed and lung cells were cultured. The concentration of IL-4, IL-5, IL-13, IFN-γ, IL-10 and IL-17 were measured by flow cytometry. Results are expressed as the mean and standard deviation for each group. *p<0,05, ** p<0,01 and *** p<0,001 by Mann Whitney test.</p

Effect of prophylactic immunization protocol with 10 µg of Der f1 DNA on respiratory function.

<p>Airway resistance and Compliance was measured at day 35 using Flexivent with instillation of 5 to 20/ml methacholine in non asthmatic non vaccinated mice (n = 6, ) Der f (n = 7, O ), pCMV-βgal mice (n = 9,) and pVAX-Der f1 (n = 9,▾) mice. Results are expressed in increased fold, as a mean for each group ± standard deviation. *p<0,05 by Mann Whitney test.</p

Cytokine concentrations (pg/ml) in vaccinated mice spleen cell culture supernatants after stimulation by medium of Der f1.

*<p>p<0,05 between pCMV-βgal and pVAX-Der f1 and ^$ p<0,05 between Der f and pVAX-Der f1.</p