An Antibody Persistent and Protective Two rSsCLP-Based Subunit Cocktail Vaccine against Sarcoptes scabiei in a Rabbit Model

Abstract

Scabies is a highly contagious disease caused by Sarcoptes scabiei which burrows into stratum corneum of host's skin. In this study, after optimizing vaccination schedule, a vaccination trial is comprised of three test groups of rabbits (n = 10/group) by immunization with (1) rSsCLP5; (2) rSsCLP12; or (3) a mixture of rSsCLP5 and rSsCLP12, three biological replicates groups (n = 10/group) and three control groups (n = 10/group). Levels of specific IgG, total IgE and cytokines in sera were detected and histopathologically analyzed as indicators of vaccine effects. The results showed that 85% (17/20) of rabbits exhibited no detectable skin lesions of S. scabiei infestation in mixed protein groups compared to single protein groups with 75% (15/20) and 70% (14/20), respectively. Moreover, the deworming rates of mixed groups are increased by 10%-20% compared with that of single groups. Each of six groups immunized with rSsCLP displayed significant increases of specific IgG, total IgE, IL-10, and TNF-α. The degree of skin damage in test groups also significantly lower than that of control groups. Thus, purified rSsCLP5 and rSsCLP12 subunit cocktail vaccine induced robust immune protection and could significantly decrease mite populations to reduce the direct transmission between rabbits.

Keywords: Sarcoptes scabiei; chitinase like-proteins; immune-protection; persistent antibody; scabies; sub-unit cocktail vaccine.

Figure 1

The collection of S. scabiei and mite challenge. (1) The skin scrapings from euthanized rabbits were collected and incubated at 38 °C; (2) the mites were counted after a 2-h incubation; (3) the mites were collected into a pipe; (4–6): rabbits were challenged via the two hind limbs.

Figure 2

Clinical symptoms in rabbits after mite challenge for four weeks. unchallenged, the rabbits free of mite infestation; PBS, vaccinated with 1 mL 0.01 M PBS each time; QuilA adjuvant, 1 mL QuilA saponin at a concentration of 1 mg/mL (dissolved in PBS) each time; Trx-His-S tag protein, vaccinated with 100 μg (first time) and 100 μg (second time) purified pET-32a (+) Trx-His-S tag protein with 1 mL QuilA saponin at a concentration of 1 mg/mL (dissolved in PBS); rSsCLP₅, rSsCLP₅’, rSsCLP₁₂, and rSsCLP₁₂ refer to the test groups immunized with 100 μg purified rSsCLP5 or rSsCLP12 protein with 1 ml Quil-A saponin at a concentration of 1 mg/mL (dissolved in PBS) for the first and second immunizations, respectively; rSsCLP₅ + rSsCLP₁₂ and rSsCLP₅ + rSsCLP₁₂’ refer to the test groups immunized with the mixture of 100 μg rSsCLP5 protein and 100 μg rSsCLP12 with 1 mL Quil-A saponin at a concentration of 1 mg/mL (dissolved in PBS) for two immunizations. The rSsCLP₅ and rSsCLP₁₂ refer to rSsCLP5 and rSsCLP12, respectively.

Figure 3

Lesion areas of the rabbit hind limbs at four weeks post-mite challenge. Group 1, vaccinated with 1 mL 0.01 M PBS each time; group 2, vaccinated with 1 mL QuilA saponin at a concentration of 1 mg/mL (dissolved in PBS) each time; group 3, vaccinated with 100 μg (first time) and 100 μg (second time) purified pET-32a (+)Trx-His-S tag protein with 1 mL QuilA saponin at a concentration of 1 mg/mL (dissolved in PBS); group 4, group 5, group 6, and group 7 refer to the test groups immunized with 100 μg purified rSsCLP5 or rSsCLP12 protein with 1 mL Quil-A saponin at a concentration of 1 mg/mL (dissolved in PBS) for the first and second immunizations, respectively; group 8 and group 9 refer to the test groups immunized with the mixture of 100 μg rSsCLP5 protein and 100 μg rSsCLP12 with 1 mL Quil-A saponin at a concentration of 1 mg/mL (dissolved in PBS) for the two immunizations. The lesion areas in the rSsCLP immunized groups (rSsCLP5, rSsCLP12, and rSsCLP5 + rSsCLP12) were significantly lower than that in the PBS control (*** p < 0.01), Quil A control (### p < 0.01) and Trx-His-S tag protein control (+++ p < 0.01).

Figure 4

Variation of specific IgG (a and b) and total IgE (c) levels in the sera of immunized rabbits. a: the OD 450 nm value of the specific IgG antibodies was detected by an rSsCLP5-based indirect ELISA; b: the OD 450 nm value of specific IgG antibodies was detected by an rSsCLP12-based indirect ELISA; c: the total IgE antibody concentration (pg/mL) was detected by an ELISA kit (Elabscience, Wuhan, China). The rSsCLP₅ and rSsCLP₁₂ refer to rSsCLP5 and rSsCLP12, respectively.

Figure 5

Variation of cytokines IL-10 (a), TNF-α (b), IL-4 (c), and IFN-γ (d) in the sera of immunized rabbits. a, the concentration of IL-10 (pg/mL) was detected by an ELISA kit (Elabscience, Wuhan, China); b, the concentration of TNF-α (pg/mL) was detected with an ELISA kit (Elabscience, Wuhan, China); c, the concentration of IL-4 (pg/mL) was detected using an ELISA kit (Elabscience, Wuhan, China); d, the concentration of IFN-γ (pg/mL) was detected using an ELISA kit (Elabscience, Wuhan, China). The rSsCLP₅ and rSsCLP₁₂ refer to rSsCLP5 and rSsCLP12, respectively.

Figure 6

Histopathology of the hind limbs of the challenged rabbits. (a) stained with hematoxylin and eosin (H&E). (b) stained with toluidine. Thick blue arrows refer to the epidermis; thick black arrows refer to crusts caused by S. scabiei; thin black arrows refer to mites in the skin. The rSsCLP₅ and rSsCLP₁₂ refer to rSsCLP5 and rSsCLP12, respectively.