A chitinase-like protein from Sarcoptes scabiei as a candidate anti-mite vaccine that contributes to immune protection in rabbits

Abstract

Background: Scabies is caused by Sarcoptes scabiei burrowing into the stratum corneum of the host's skin and is detrimental to the health of humans and animals. Vaccines are an attractive alternative to replace the acaricides currently used in their control.

Methods: In the present study, the S. scabiei chitinase-like protein 5 (SsCLP5) was characterized and recombinant SsCLP5 (rSsCLP5) was evaluated as a candidate vaccine protein for anti-mite protection in rabbits. The expression, characterization and immunolocalization of SsCLP5 were examined. Vaccination experiments were performed on three test groups (n = 12 per group) immunized with purified rSsCLP5. Control groups (n = 12 per group) were immunized with PBS, QuilA saponin or empty vector protein. After challenge, the inflammatory reaction and skin lesions were graded and rSsCLP5 indirect ELISA was used to detect antibody IgG levels in serum samples at the time of vaccination and post-challenge.

Results: The results showed that rSsCLP5 had high immunoreactivity and immunogenicity. In S. scabiei, SsCLP5 had a wide distribution in the chewing mouthpart, legs and exoskeleton, especially the outer layer of the exoskeleton. Vaccination with rSsCLP5 resulted in 74.3% (26/35) of rabbits showing no detectable lesions after challenge with S. scabiei.

Conclusions: Our data demonstrate that rSsCLP5 is a promising candidate for a recombinant protein-based vaccine against S. scabiei. This study also provides a method for studying scabies vaccine using rabbit as an animal model and a basis for screening more effective candidate proteins.

Keywords: Chitinase-like protein 5; Recombinant protein; Sarcoptes scabiei; Scabies; Vaccine.

Fig. 1

Phylogenetic relationships between SsCLPs and CLPs and chitinases from other species. The unrooted phylogenetic tree was constructed using CLP and chitinase sequences from approximately 40 species by the maximum likelihood (ML) method in MEGA software. CLP sequences or chitinase sequences were used in the tree (with their GenBank or SwissProt accession numbers). The number behind the protein acronym (for example Chi1, CLP2, CLP3, etc.) is from the NCBI database which represents more than one kind of protein in the species. Accession numbers are as follows: Stegodyphus mimosarum-Chi2 (gb: KFM81240.1); Parasteatoda tepidariorum-CLP1 (gb: XP_015906877.1); Limulus polyphemus-CLP1 (gb: XP_013781598.1); S. scabiei-CLP11 (gb: KPM07813.1); Tetranychus cinnabarinus-Chi1 (gb: AQX83302.1); Tetranychus urticae-Chi1 (gb: XP_015789865.1); S. scabiei-CLP8 (gb: KPM09395.1); E. maynei-CLP (gb: OTF83884.1); Nilaparvata lugens-Chi2 (gb: XP_022199541.1); Bactrocera dorsalis-Chi (gb: AEN03034.1); S. scabiei-CLP4 (gb: KPM08718.1); B. dorsalis-Chi8 (gb: AQR60114.1); Drosophila willistoni-CLP1 (gb: XP_002074824.1); Copidosoma floridanum-CLP1 (gb: XP_014210861.1); Aedes aegypti-CLP (gb: EAT45692.1); Melipona quadrifasciata-Chi1 (gb: KOX77106.1); Bombus terrestris-Chi1 (gb: XP_003395393.1); S. scabiei-CLP6 (gb: KPM08912.1); Homarus americanus-Chi (gb: ABQ59095.1); T. cinnabarinus-Chi (gb: APA05224.1); T. urticae-Chi3 (gb: XP_015795759.1); S. scabiei-CLP12 (gb: KPM07848.1); Haemaphysalis longicornis-Chi (gb: BAC06447.1); Anopheles sinensis-CLP (gb: KFB36184.1); N. lugens-Chi (gb: AJO25042.1); Papilio xuthus-Chi3 (gb: KPJ02577.1); Bombyx mori-Chi10 (gb: XP_021203926.1); Drosophila grimshawi-Chi (gb: XP_001991892.1); Drosophila melanogaster-Chi (gb: NP_001245599.1); Zootermopsis nevadensis-CLP1 (gb: XP_021912899.1); T. cinnabarinus-CLP1 (gb: ARN16744.1); S. scabiei-CLP1 (gb: KPM05982.1); Nephila clavipes-CLP1 (gb: PRD36178.1); Ixodes scapularis-Chi (gb: XP_002403819.1); S. scabiei-CLP2 (gb: KPM07295.1); E. maynei-CLP (gb: OTF69711.1) ; Spodoptera litura-CLP1 (gb: XP_022814601.1); S. scabiei-CLP9 (gb: KPM10408.1); E. maynei-CLP (gb:OTF70345.1); I. scapularis-Chi (gb: XP_002434337.1); Cimex lectularius-CLP (gb: XP_014241842.1); P. tepidariorum-CLP (gb: XP_015916511.1); S. mimosarum-Chi (gb: KFM61851.1); S. scabiei-CLP3 (gb: KPM07498.1 ); E. maynei-CLP (gb: OTF83991.1); S. scabiei-CLP10 (gb: KPM11497.1); E. maynei-CLP (gb: OTF77275.1); T. urticae-CLP (gb: XP_015790837.1); P. xuthus-Chi1 (gb: KPI93103.1); N. lugens-Chi (gb: AJO25038.1); Harpegnathos saltator-Chi (gb: EFN85614.1); Camponotus floridanus-CLP (gb: EFN63520.1); Struthio camelus australis-CDCP1 (gb: KFV81001.1 ) CDCP1; Egretta garzetta-CDCP1 (gb: KFP09506.1); Oryzias melastigma-CDCP (gb: XP_024137025.1); S. scabiei-CLP5 (gb: KPM08736.1); E. maynei-CLP (gb: OTF80145.1); L. polyphemus-CDCP (gb: XP_013787202.1); Daphnia magna-CDCP (gb: KZS04185.1); S. scabiei-CLP7 (gb: KPM08913.1) and E. maynei-CLP (gb: OTF84112.1). Abbreviations: gb, GenBank or SwissProt accession numbers; Chi, chitinase; CLP, chitinase-like protein; CDCP, chitinase domain-containing protein

Fig. 2

SDS-PAGE and western blotting of SsCLP5. Lane M: protein molecular weight markers (in kDa); Lane 1: pet-32a (+) expression vector protein; Lane 2: non-purified rSsCLP5 [soluble protein expressed from Escherichia coli BL21 (DE3)]; Lane 3: purified rSsCLP5; Lane 4: total crude proteins from S. scabiei; Lane 5: purified rSsCLP5 detected by serum (diluted 1:120 with 0.01 M PBS) from a rabbit naturally infested with S. scabiei (experimental group); Lane 6: purified rSsCLP5 detected by serum (diluted 1:120 with 0.01 M PBS) from a rSsCLP5-vaccinated rabbit (positive control); Lane 7: purified rSsCLP5 detected by pre-immune rabbit serum (diluted 1:120 with 0.01 M PBS; negative control); Lane 8: total crude proteins detected with rabbit anti-rSsCLP5 serum (diluted 1:120 with 0.01 M PBS). Samples derived from the same experiment and gels/blots were processed in parallel

Fig. 3

Immunolocalization of SsCLP5 in S. scabiei tissue (a, b) and H & E sections of S. scabiei. a S. scabiei incubated with anti-rSsCLP5 antibody as the primary antibody. b Negative control (antibody of the pre-immune rabbit serum), c H & E sections of S. scabiei. Abbreviations: A, anterior end of mite; P, posterior end of mite; IT, integument of the exoskeleton; M, mouthparts; L, legs; SP, spicules; SB, stomach blocks

Fig. 4

Clinical symptoms in rabbits before, two and four weeks post-challenge. Series a shows normal hind limbs in rabbits before challenge, series b shows clinical symptoms of hind limbs in rabbits after challenge with scabies mites for two weeks, and series c shows clinical symptoms of hind limbs in rabbits after challenge with scabies mites for four weeks. Key: PBS, immunized with 1 ml 0.01 M PBS each time; Quil A, immunized with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS) each time; vector protein, immunized with 100 μg (first time) and 200 μg (second time) purified pET32a(+) vector protein with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS); rSsCLP5, rSsCLP5’, and rSsCLP5” refer to the test groups immunized with 100 μg then 200 μg purified rSsCLP5 protein with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS) at the first and second immunizations

Fig. 5

Inflammatory reaction grades and skin lesions at two (a) and four weeks (b) post-challenge. The colored lines indicate the group average. Key: PBS, immunized with 1 ml 0.01 M PBS each time; Quil A, immunized with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS) each time; vector protein, immunized with 100 μg (first time) and 200 μg (second time) purified pET32a(+) vector protein with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS); rSsCLP5, rSsCLP5’, and rSsCLP5” refer to the test groups immunized with 100 μg then 200 μg purified rSsCLP5 protein with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS) at the first and second immunizations. The grades of inflammatory reaction and skin lesions in the rSsCLP5 immunized groups (rSsCLP5, rSsCLP5’, and rSsCLP5”) were significantly lower compared to the control groups at week 2 post-challenge (F_(5,63) = 13.575, P < 0.0001) and at week 4 post-challenge (F_(5,63) = 23.38, P < 0.0001)

Fig. 6

Area of lesions on rabbit hind limbs at four weeks post-challenge. The colored lines indicate the group average. Key: PBS, immunized with 1 ml 0.01 M PBS each time; Quil A, immunized with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS) each time; vector protein, immunized with 100 μg (first time) and 200 μg (second time) purified pET32a(+) vector protein with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS); rSsCLP5, rSsCLP5’, and rSsCLP5” refer to the test groups immunized with 100 μg then 200 μg purified rSsCLP5 protein with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS) at the first and second immunizations. The values of area of lesions in the rSsCLP5 immunized groups (rSsCLP5, rSsCLP5’, and rSsCLP5”) were significantly lower compared to PBS control, Quil A control and vector protein control at week 4 post-challenge (F_(5,63) = 36.99, P < 0.0001)

Fig. 7

Weight changes of the rabbits at week 4 post-challenge compared to prior immunization. The colored lines indicate the group average. Key: PBS, immunized with 1 ml 0.01 M PBS each time; Quil A, immunized with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS) each time; vector protein, immunized with 100 μg (first time) and 200 μg (second time) purified pET32a(+) vector protein with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS); rSsCLP5, rSsCLP5’, and rSsCLP5” refer to the test groups immunized with 100 μg then 200 μg purified rSsCLP protein with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS) at the first and second immunizations. The weight changes of rabbits in the rSsCLP5 immunized groups (rSsCLP5 and rSsCLP5’) were significantly higher compared to PBS control, Quil A control and vector protein control at week 4 post-challenge (F_(5,63) = 2.689, P = 0.029)

Fig. 8

Mite burden in rabbits at week 4 post-challenge. Key: PBS, immunized with 1 ml 0.01 M PBS each time; Quil A, immunized with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS) each time; vector protein, immunized with 100 μg (first time) and 200 μg (second time) purified pET32a(+) vector protein with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS); rSsCLP5, rSsCLP5’, and rSsCLP5” refer to the test groups immunized with 100 μg then 200 μg purified rSsCLP protein with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS) at the first and second immunizations. Data columns correspond to the mean values; error bars represent the standard error. The mean values of mite burden in test groups immunized with rSsCLP5 were significantly lower compared to the control groups at week 4 post-challenge (F_(5,63) = 39.354, P < 0.0001)

Fig. 9

Variation of specific IgG antibody levels in sera of immunized rabbits as detected by ELISA. Rabbits were immunized twice. Key: PBS, immunized with 1 ml 0.01 M PBS each time; Quil A, immunized with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS) each time; vector protein, immunized with 100 μg (first time) and 200 μg (second time) purified pET32a(+) vector protein with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS); rSsCLP5, rSsCLP5’, and rSsCLP5” refer to the test groups immunized with 100 μg then 200 μg purified rSsCLP5 protein with 1 ml QuilA saponin at concentration of 1 mg/ml (dissolved in PBS) at the first and second immunizations. Data points correspond to the mean values; error bars represent the standard error. The groups immunized with the rSsCLP5 had significantly higher IgG levels compared to the control groups (F_(5,63) = 37.285, P < 0.0001) from week 1 after first immunization to the end of the experiment