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

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.

Tumor Susceptibility Gene 101 (TSG101) Contributes to Virion Formation of Porcine Reproductive and Respiratory Syndrome Virus via Interaction with the Nucleocapsid (N) Protein along with the Early Secretory Pathway

“…Mouse anti-PRRSV N and GP5 antibodies were kept in our laboratory ( 71 ). The PRRSV Nsp9 nanobody containing porcine IgG Fc (Nb6-pFc) was kindly provided by Qin Zhao from Northwest A&F University ( 72 ).…”

Highly pathogenic PRRSV upregulates IL-13 production through nonstructural protein 9–mediated inhibition of N6-methyladenosine demethylase FTO

“…Furthermore, 551 to 600 aa in nsp9 were found to play important roles in the stimulation of IL-13 production by reducing m 6 A demethylase FTO expression. Recent studies have shown that amino acids at positions 519, 544, 586, 588, 590, 592, 611, and 643 in nsp9 regulated the high virulence of PRRSV, interacted with other viral proteins, enhanced pathogenicity, and controlled cellular immune response ( 65 , 71 , 72 , 73 ). The amino acid mutations at positions 519 and 544 of PRRSV nsp9 were found to be necessary for the rescue of PRRSV and critical to the replication efficiency of HP-PRRSV, contributing to enhanced pathogenicity ( 73 ).…”

“…The amino acids at positions 586 and 592 in nsp9 have been reported to contribute to the replication efficiency of the Chinese HP-PRRSV in PAMs and act as critical sites determining the viral virulence in piglets ( 71 ). The amino acid residues at positions 588, 590, and 643 in nsp9 and at positions 62, 105, and 107 in nanobodies 6 (nb6) have been observed to be involved in nsp9–nb6 interaction, contributing to the inhibition of PRRSV replication ( 72 ). The E608 and E611 aa in nsp9 and Q85 aa in the N protein have been noted to be the pivotal residues involved in the N–nsp9 interaction, acting as anti-attenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis ( 65 ).…”

A Novel Blocking Enzyme-Linked Immunosorbent Assay Based on a Biotinylated Nanobody for the Rapid and Sensitive Clinical Detection of Classical Swine Fever Virus Antibodies

“…Several studies have shown that Nbs can inhibit virus replication ( 36 – 38 ). E2 is the most immunogenic protein of CSFV and can produce clinical protective antibodies against CSFV challenge ( 1 ).…”