Nanobody Nb6 fused with porcine IgG Fc as the delivering tag to inhibit porcine reproductive and respiratory syndrome virus replication in porcine alveolar macrophages

Lu Zhang,En-Min Zhou,Guixi Zhang,Kaissar Tabynov,Huanhuan Lv,Lizhen Wang,Jingjing Huang,Qin Zhao,Shuaishuai Cao

doi:10.1186/s13567-020-00868-9

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious virus that has led to enormous economic loss worldwide because of ineffective prevention and treatment. In view of their minimized size, high target specificity and affinity, nanobodies have been extensively investigated as diagnostic tools and treatments of many diseases. Previously, a PRRSV Nsp9-specific nanobody (Nb6) was identified as a PRRSV replication inhibitor. When it was fused with cell-penetrating peptide (CPP) TAT, Nb6-TAT could enter the cells for PRRSV suppression. However, delivery of molecules by CPP lack cell specificity and have a short duration of action. PRRSV has a tropism for monocyte/macrophage lineage, which expresses high levels of Fcγ receptors. Herein, we designed a nanobody containing porcine IgG Fc (Fcγ) to inhibit PRRSV replication in PRRSV permissive cells. Fcγ fused Nb6 chimeric antibody (Nb6-pFc) was assembled into a dimer with interchain disulfide bonds and expressed in a Pichia pastoris system. The results show that Nb6-pFc exhibits a well-binding ability to recombinant Nsp9 or PRRSV-encoded Nsp9 and that FcγR-mediated endocytosis of Nb6-pFc into porcine alveolar macrophages (PAM) was in a dose-dependent manner. Nb6-pFc can inhibit PRRSV infection efficiently not only by binding with Nsp9 but also by upregulating proinflammatory cytokine production in PAM. Together, this study proposes the design of a porcine IgG Fc-fused nanobody that can enter PRRSV susceptible PAM via FcγR-mediated endocytosis and inhibit PRRSV replication. This research reveals that nanobody-Fcγ chimeric antibodies might be effective for the control and prevention of monocyte/macrophage lineage susceptible pathogeneses.

Highlights

Antibodies represent the largest and fastest growing class of drugs against numerous diseases in the pharmaceutical industry
The Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) Nsp9 in infected MARC-145 cells was pulled down by the Nb6-pFc but not by Nb53-pFc (Figure 2C). These results indicate that Nb6-pFc expressed by Pichia pastoris can interact with PRRSV Nsp9
We suggest that Nbs-pFc treatment can induce porcine alveolar macrophages (PAM) activation and upregulate cytokine expression, contributing to the inhibition of PRRSV infection of PAM at early stages

Summary

Introduction

Antibodies represent the largest and fastest growing class of drugs against numerous diseases in the pharmaceutical industry. Nbs carrying different tags can enter target cells, making them suitable for the treatment of the diseases [4]. While many works on the production and design of Nbs for treatment of human diseases are currently available [5], there are limited studies on the preparation of Nbs against animal diseases. Previous studies documented that Nsp is a relatively conserved region in the PRRSV genome [19] and an ideal target of antiviral design for the control of PRRSV infection. We produced Nb6 fused with trans-activating transduction (TAT) peptide and found that it can inhibit replication of two genotypes of PRRSV isolates in MARC-145 cells and PAM [10, 20]. TAT exhibits several disadvantages including a short half-life in blood and nonspecific delivery to normal tissue [21]

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Discussion

Conclusion