Intranasally administered antigen 85B gene vaccine in non-replicating human Parainfluenza type 2 virus vector ameliorates mouse atopic dermatitis

Abstract

Atopic dermatitis (AD) is a refractory and recurrent inflammatory skin disease. Various factors including heredity, environmental agent, innate and acquired immunity, and skin barrier function participate in the pathogenesis of AD. T -helper (Th) 2-dominant immunological milieu has been suggested in the acute phase of AD. Antigen 85B (Ag85B) is a 30-kDa secretory protein well conserved in Mycobacterium species. Ag85B has strong Th1-type cytokine inducing activity, and is expected to ameliorate Th2 condition in allergic disease. To perform Ag85B function in vivo, effective and less invasive vaccination method is required. Recently, we have established a novel functional virus vector; recombinant human parainfluenza type 2 virus vector (rhPIV2): highly expressive, replication-deficient, and very low-pathogenic vector. In this study, we investigated the efficacy of rhPIV2 engineered to express Ag85B (rhPIV2/Ag85B) in a mouse AD model induced by repeated oxazolone (OX) challenge. Ear swelling, dermal cell infiltrations and serum IgE level were significantly suppressed in the rhPIV2/Ag85B treated mouse group accompanied with elevated IFN-γ and IL-10 mRNA expressions, and suppressed IL-4, TNF-α and MIP-2 mRNA expressions. The treated mice showed no clinical symptom of croup or systemic adverse reactions. The respiratory tract epithelium captured rhPIV2 effectively without remarkable cytotoxic effects. These results suggested that rhPIV2/Ag85B might be a potent therapeutic tool to control allergic disorders.

Figure 1. Schematic diagram of constructs and strategy used in this study.

A. The constructs of recombinant hPIV2/EGFP and hPIV2/Ag85B. The EGFP or Ag85B gene open reading frame was engineered to be flanked by hPIV2-specific gene end of NP gene (R2), intergenic sequence (IG), and gene start (R1) transcriptional signal of V/P gene. It was inserted into a cloned cDNA of the hPIV2 antigenome at a Not I site that had been engineered to be at 5′-noncoding region of NP gene. A genomic nucleotide length divisible by six (the rule of six) was maintained. For generating of replication-deficient virus, two stop codons (▾) were introduced on the M gene. B. Schedule for the development of a hapten-induced atopic dermatitis model and vaccination of rhPIV2/Ag85B. Mice were initially sensitized with 20 µl of 0.5% OX solution to their right ear 7 days prior to the first challenge (day -7) and then 20 µl of 0.5% OX solution was repeatedly applied on the right ear 3 times per week from day 0 until day 21. Mice were inoculated intranasally with 20 µl (5×10⁶ TCID₅₀) of rhPIV2/Ag85B or rhPIV2 on day 4. rhPIV2 vector or phosphate buffered saline (PBS) were also applied as controls. On day 16, mice were vaccinated again intranasally or subcutaneously with PBS, rhPIV2 or rhPIV2/Ag85B. C. Summarized schedule of the experimental groups.

Figure 2. Expression of EGFP from rhPIV2/EGFP.

A. HaCat cells were infected with rhPIV2/EGFP at an MOI of 0.5. Three days after, EGFP was clearly visualized using a fluorescence microscopy (x100). B. The rhPIV2/EGFP (5×10⁶ TCID₅₀) were administered to a wild type BALB/c mice intranasally EGFP was visualized clearly in the airway epithelial cells 4 days after administration (x200, upper right box, x400).

Figure 3. Anti-inflammatory effects of vaccination with rhPIV2/Ag85B.

A. Clinical manifestation of the ear skin at 6 hours after OX challenge on day 21. The control groups (PBS, PIV2 ear, and PIV2 nasal on the panel) showed severe edema with erythema, however the intranasal and/or subcutaneous administration of the rhPIV2/Ag85B (Ag nasal and Ag ear on the panel, respectively) clearly reduced skin reactions in OX-sensitized mice. B. Ear thickness measured before and 6 hours after each OX application on day 21. The ear swelling was suppressed significantly in rhPIV2/Ag85B treated groups in two ways compared to those in the placebo treated groups. (*P<0.05, **P<0.01.) C. Histopathological changes of the ear skin obtained on day 21 in paraffin embedded sections stained with hematoxylin and eosin. The placebo treated groups (PBS, PIV2 ear and PIV2 nasal on the panel) revealed marked inflammatory reactions with acanthosis and ulceration in epidermis, and marked edema with cellular infiltration including mononuclear cells and neutrophils in the dermis. The skin infiltration of inflammatory cells and epidermal thickness were decreased in rhPIV2/Ag85B treated group (Ag85B ear and Ag85B nasal on the panel). Original magnification × 100. D. Plasma IgE levels on day 21. Plasma IgE level was decreased in rhPIV2/Ag85B treated groups (Ag ear and Ag nasal). *P<0.05, **P<0.01.

Figure 4. Changes in cytokines mRNA expression levels in the ears of AD mice by vaccination of rhPIV2/Ag85B.

Cytokines: IL-4 (panel A), IFN-γ (panel B), IL-10 (panel C), TGF-β (panel D), TNF-α (panel E), MIP2-α (panel F), IL-2 (panel G), IL-17 (panel H), mRNA expression in the ear lesions measured with Quantitative RT-PCR. Expressions of IL-4, TNF-α and MIP2-α mRNA were significantly decreased in the ear skin treated with intra-nasally rhPIV2/Ag85B treated group compared to those of control groups. Meanwhile, the expression levels of mRNA of IFN-γ, IL-10, TGF-β and IL-2 were significantly elevated in rhPIV2/Ag85B intra-nasally treated group compared to those of control groups. *P<0.05, **P<0.01, ***P<0.001.

Figure 5. Immunostaining for Tregs in the inflamed ear skin lesions.

A. CD4⁺ T cells are displayed with green fluorescence (1), and Foxp3⁺ T cells are with red (2). Merged yellow color means Foxp3⁺CD4⁺ T cells (3) (x100). B. The number of skin infiltrating CD4⁺ T cells is less in Ag nasal group and Ag ear group compared to that of PBS-treated group. Although it does not reach statistical significance, CD4⁺ T cell number is less in Ag nasal group compared to that of Ag ear group. C. The number of Foxp3⁺CD4⁺ T cells in the inflamed ear skin is significantly increased in both of the intra-nasal and ear-subcutaneous rhPIV2/Ag85B application groups.