Characterization and evaluation of a Sarcoptes scabiei allergen as a candidate vaccine

Abstract

Background: Sarcoptic mange caused by the mite Sarcoptes scabiei is a worldwide disease affecting both humans and animals. Here we report the molecular characterization and evaluation of a recombinant S. scabiei tropomyosin (SsTm) protein in a vaccination trial in rabbits.

Methods: The full-length cDNA was cloned in a bacterial pET vector, and the recombinant protein was expressed in BL21 (DE3) cells and purified. Using specific rabbit antiserum, tropomyosin was localized immunohistochemically in mite tissue sections. Vaccination trials with the recombiant SsTm was carried out in New Zealand rabbits.

Results: The full-length open reading frame (ORF) of the 852 bp cloned gene from S. scabiei encodes a 32.9 kDa protein. The amino acid sequence showed 98.94%, 97.89% and 98.59% homology to Dermatophagoides farina and Dermatophagoides pteronyssinus group 10 allergens and Psoroptes ovis tropomyosin, respectively. Tropomyosin was localized immunohistochemically in mite tissue sections mainly in the mouthparts, legs and integument of the epidermis. The predicted cross-reactivity of SsTm indicated that it is an allergenic protein. While vaccination with the recombiant SsTm resulted in high levels of specific IgG (P < 0.01), a low IgE antibody response and no significant protection against S. scabiei challenge were observed. After challenge, specific IgG levels remained significantly higher than the control (P < 0.01), while changes of total IgE levels were not significant (P > 0.05). However, the lesion areas in the vaccination group decreased at the end of the experiment compared with controls.

Conclusions: Although vaccination with recombinant SsTm did not efficiently control sarcoptic mange in rabbits, the immunogenic properties of tropomyosin suggest it may be developed as a vaccine with alternative adjuvants or delivery methods.

Figure 1

Neighbor-joining phylogenetic tree of selected tropomyosin proteins. GenBank accession numbers for each sequence are as follows: Homarus americanus, AF034954; Haemaphysalis longicornis, AF534184; Dermatophagoides pteronyssinus, D17682; Dermatophagoides gallinae, AM167555; Schistosoma japonicum, L76203; Boophilus micoplus, AF124514; Psoroptes ovis, AM114276; Anisakis simplex, Y19221; Lepidoglyphus destructor, AJ250096; Haliotis asinina, AY320360; Sarcoptes scabiei, JF922117. The percentage of support from 1000 bootstrap replicates is indicated at the nodes. Phylogenetic analysis was conducted using MEGA 4.0 [25].

Figure 2

Comparison of SsTm with the tropomyosin amino acid sequences of other species. Genebank accession numbers: A. simplex, CAB93501; B. microplus, AAD17324; Der f 10, BAA04557; Der p 10, CAJ44440; L. destructor, CAB71342; P. ovis, CAJ38272; S. scabiei, JF922117.

Figure 3

Western blotting analysis of recombinant proteins. M, protein marker; lane 1, purified recombinant proteins; lane 2, Western blot results.

Figure 4

Immunolocalization of tropomyosin in sections of S. scabiei.  (A, B) Staining with anti-tropomyosin as primary antibody. (C) Control (pre-immune sera). The EnVision TM + System-HRP(DAB) (DAKO) was used, according to the manufacturer’s instructions for detection of the rabbit antibodies. M, mouthparts; L, legs; S, stomach blocks; IE, the integument of epidermis; A, anterior end of mite; P, posterior end of mite.

Figure 5

Specific IgG(A) and IgE(B) antibody levels in sera of rabbits immunized with S.  scabiei tropomyosin detected by ELISA. Rabbits were vaccinated four times with SsTm, QuilA and saline and challenged with approximately 2000 mites. Results are shown as means (± S.D.). OD values were determined as absorbance at 450 nm. V1, first vaccination; V2, second vaccination; V3, third vaccination; V4, fourth vaccination. *P < 0.01, compared with NS control group.