Generation and functional analysis of single chain variable fragments (scFvs) targeting the nucleocapsid protein of Porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, which can cause death in suckling piglets. Vaccines confer only partial protection against new mutant strains, whereas antibodies targeting virus-encoded proteins may be effective prophylactics. In this study, we constructed a recombinant single chain variable fragment (scFv) library from the spleens of two pigs immunized with a recombinant PEDV nucleocapsid (N) protein. Among the positive clones directed against PEDV N protein isolated from the library, four scFvs that showed higher affinity for N were functionally analyzed. These scFvs specifically bound to the PEDV N protein, but not to the transmissible gastroenteritis virus (TGEV) N protein. Their framework regions were highly conserved, whereas their complementarity-determining regions displayed clear diversity. An immunofluorescence assay showed the co-localization of the four scFvs with PEDV N protein in cells. They significantly suppressed PEDV replication, detected with reverse transcription (RT)-quantitative PCR (qPCR; P < 0.01). Two of them significantly reduced the viral titer at 48 hpi and 72 hpi (P < 0.05). In addition, they observably suppressed the production of viral protein at 72 hpi. The expression of interferons, interferon regulatory factor 3 (IRF3), and IRF7 was assessed with RT-qPCR, which indicated that PEDV dramatically suppressed the transcription of interferon-λ1 and IRF7 and that the scFvs significantly upregulated their expression (P < 0.05). These findings facilitated the investigation of the mechanism by which PEDV evaded the host immune response and suggested that these porcine scFvs were potential candidate agents for the prevention and treatment of porcine diarrhea caused by PEDV. KEY POINTS: • Four scFvs targeting PEDV N protein were generated from porcine spleens • These scFvs co-localized with PEDV N protein and suppressed PEDV replication • These scFvs significantly upregulated IFN-λ1 expression.

Single-Chain Fragment Variables Targeting Leukocidin ED Can Alleviate the Inflammation of Staphylococcus aureus-Induced Mastitis in Mice.

Staphylococcus aureus is a vital bovine mastitis pathogen causing huge economic losses to the dairy industry worldwide. In our previous studies, leukotoxin ED (LukED) was detected in most S. aureus strains isolated from bovine mastitis. Here, four single-chain fragment variables (scFvs) (ZL8 and ZL42 targeting LukE, ZL22 and ZL23 targeting LukD) were obtained using purified LukE and LukD proteins as the antigens after five rounds of bio-panning. The complementarity-determining region 3 (CDR3) of the VH domain of these scFvs exhibited significant diversities. In vitro, the scFvs significantly decreased LukED-induced cell killing by inhibiting the binding of LukED to chemokine receptors (CCR5 and CXCR2) and reduced the death rates of bovine neutrophils and MAC-T cells caused by LukED and S. aureus (p < 0.05). In an S. aureus-induced mouse mastitis model, histopathology and MPO results revealed that scFvs ameliorated the histopathological damages and reduced the infiltration of inflammatory cells (p < 0.05). The ELISA and qPCR assays showed that scFvs reduced the transcription and expression levels of Tumor Necrosis Factor-alpha (TNF-α), interleukin-1ß (IL-1ß), IL-6, IL-8 and IL-18 (p < 0.05). The overall results demonstrated the protective anti-inflammatory effect of scFvs in vitro and in vivo, enlightening the potential role of scFvs in the prevention and treatment of S. aureus-induced mastitis.

Correction: Zhang et al. Anti-Staphylococcus aureus Single-Chain Fragment Variables Play a Protective Anti-Inflammatory Role In Vitro and In Vivo. Vaccines 2021, 9, 1300.

In the original publication [...].

Anti-Staphylococcus aureus Single-Chain Fragment Variables Play a Protective Anti-Inflammatory Role In Vitro and In Vivo.

Staphylococcus aureus is a causative agent of bovine mastitis, capable of causing significant economic losses to the dairy industry worldwide. This study focuses on obtaining single-chain fragment variables (scFvs) against the virulence factors of S. aureus and evaluates the protective effect of scFvs on bovine mammary epithelial (MAC-T) cells and mice mammary gland tissues infected by S. aureus. After five rounds of bio-panning, four scFvs targeting four virulence factors of S. aureus were obtained. The complementarity-determining regions (CDRs) of these scFvs exhibited significant diversities, especially CDR3 of the VH domain. In vitro, each of scFvs was capable of inhibiting S. aureus growth and reducing the damage of MAC-T cells infected by S. aureus. Preincubation of MAC-T cells with scFvs could significantly attenuate the effect of apoptosis and necrosis compared with the negative control group. In vivo, the qPCR and ELISA results demonstrated that scFvs reduced the transcription and expression of Tumor Necrosis Factor alpha (TNF-α), interleukin-1ß (IL-1ß), IL-6, IL-8, and IL-18. Histopathology and myeloperoxidase (MPO) results showed that scFvs ameliorated the histopathological damages and reduced the inflammatory cells infiltration. The overall results demonstrated the positive anti-inflammatory effect of scFvs, revealing the potential role of scFvs in the prevention and treatment of S. aureus infections.