Soluble form of canine transferrin receptor inhibits canine parvovirus infection in vitro and in vivo

Abstract

Canine parvovirus (CPV) disease is an acute, highly infectious disease threatening the dog-raising industry. So far there are no effective therapeutic strategies to control this disease. Although the canine transferrin receptor (TfR) was identified as a receptor for CPV infection, whether extracellular domain of TfR (called soluble TfR (sTfR)) possesses anti-CPV activities remains elusive. Here, we used the recombinant sTfR prepared from HEK293T cells with codon-optimized gene structure to investigate its anti-CPV activity both in vitro and in vivo. Our results indicated that codon optimization could significantly improve sTfR expression in HEK293T cells. The prepared recombinant sTfR possessed a binding activity to both CPV and CPV VP2 capsid proteins and significantly inhibited CPV infection of cultured feline F81 cells and decreased the mortality of CPV-infected dogs, which indicates that the sTfR has the anti-CPV activity both in vitro and in vivo.

Figure 1

Structures of sTfR expression vectors and identification of sTfR expression. (a) The wild-type or codon-optimized sTfR gene expression vectors, pcDNA3.1A-CD5-sTfRw/His and pcDNA3.1A-CD5-sTfRopt/His, in which sTfR gene driven by CMV promoter was fused by human CD5 signal peptide sequence at 5′-terminus and 6 × His-tag at 3′-terminus. (b) Identification of sTfR with SDS-PAGE: lane M, protein markers; lane 1, purified sTfR. (c) Identification of sTfR with Western blot: lane M, prestained protein markers; lane 1, sTfR protein expressed from pcDNA3.1A-CD5-sTfRopt/His-transfected cells; lane 2, negative control from empty vector-transfected cells.

Figure 2

The effect of codon optimization on sTfR expression. (a) SDS-PAGE analysis for purified sTfR proteins. Lane 1, sTfR protein expressed with codon-optimized construct; lane 2, sTfR protein expressed with wild-type construct; M, protein marker. (b) Comparison of the protein expression levels with the different sTfR gene constructs.

Figure 3

The recombinant sTfR binding activities to VP2 and CPV. Two 96-well plates were coated with VP2 (1 μg/mL) (a) and CPV (1 × 10⁻⁵ TCID₅₀/mL) (b), respectively. The different amounts of sTfR (5, 2.5, 1.25, 0.63, 0.31, and 0.16 μg/mL) were added for each well (BSA as a negative control). Other two 96-well plates were also coated with VP2 (1 μg/mL) (c) and CPV (1 × 10⁻⁵ TCID₅₀/mL) (d) and were added rabbit anti-VP2 polyclonal antibodies at different amount (1, 2, 5, 10 μg/mL) for antibody blocking test. Then the TfR (2.5 μg/mL) was added for all of the wells except mock control well. The mouse anti-His antibody and goat anti-mouse IgG-AP were used for detection of the bound sTfR fused with His-tag. After development with 4-nitrophenyl phosphate substrate, the optical density (OD) for each well was measured using microplate reader at 405 nm. The sTfR binding activity was indicated by OD values.

Figure 4

Effects of sTfR on F81 cell morphology and viral tilters after CPV infection. (a) Effects of sTfR on F81 cell morphology: CPV (1 × 10⁵ TCID₅₀/mL) was incubated with same volume of 10 μg/mL sTfR or 10 μg/mL BSA (control). F81 cells seeded into 96-well plate at the density of 1 × 10⁵ cells/mL (50 μL). The sTfR-incubated or BSA-incubated (as a control) CPV was added to the well and incubated at 37°C for 6 h. Unbound viruses were removed by extensive washing with DMEM. The cells were continuously cultured until CPE occurred. Figure 4(a) shows the morphology of F81 cells infected with BSA-incubated CPV (left) and sTfR-incubated CPV (right) at 48 h postinfection, respectively. (b) Effects of sTfR on CPV titer in F81 cells: F81 cells were infected by CPV preincubated with different amounts of sTfR and BSA. The infected cells were harvested at 48 h postinfection. The virus particles were released from the cells by the repeated freezing/shawing. The virus titer was estimated by TCID₅₀.

Figure 5

The effects of sTfR treatments on the morbidity (a) and mortality (b) rates of CPV-infected dogs. The dogs were treated with sTfR before CPV challenge (sTfR + CPV) and after CPV challenge (CPV + sTfR). CPV + BSA indicates the control without sTfR treatment.