背景:猪繁殖与呼吸综合征病毒(PRRSV)是一种流行的猪病原体,近30年来,这对全球养猪业造成了不利影响。然而,由于病毒引起的免疫抑制和PRRSV的遗传多样性,目前还没有成功开发针对病毒的广泛中和策略。抗病毒肽和纳米抗体以其易于生产和在实践中的功效引起了广泛的关注。在这项研究中,通过将PRRSV特异性非中和纳米抗体与靶向PRRSV蛋白的受体结合域(RBD)的CD163衍生肽组合,开发了四种新的融合蛋白,称为纳米抗体肽缀合物(NPC)。
结果:使用两个纳米抗体分别针对PRRSVN和nsp9成功构建了四个NPC,分别与来自猪CD163的两种抗病毒肽4H7或8H2重组。所有四种NPC均表现出与PRRSV结合的特异性能力以及以剂量依赖性方式对PRRSV的各种谱系的广泛抑制作用。NPC在Nb组分的辅助下通过CD163表位肽干扰PRRSV蛋白的RBD与PRRSV预附着阶段中的CD163的结合。NPC还在附着后阶段抑制病毒复制,抑制作用取决于NPCs中Nb部分的抗病毒功能,包括在长病毒RNA合成中的干扰,NF-κB和IFN-β激活。此外,预测了NPC-N/nsp9-4H7的中和结构域4H7的aaK31和T32位点与PRRSVGP2a的基序171NLRLTG176之间的相互作用。NPC-N/nsp9-8H2的中和结构域8H2的基序28SSS30也可以形成氢与PRRSVGP3的基序152NAFLP156结合。该研究为PRRSV蛋白的RBD的结构特征和潜在功能意义提供了有价值的见解。最后,如小鼠模型所示,体内鼻内接种12-24小时的NPC维持针对PRRSV的显著中和活性。这些发现激发了NPC作为预防措施的潜力,可以降低宿主人群针对PRRSV等呼吸道感染因子的传播风险。
结论:本研究的目的是开发基于肽的生物活性化合物以中和各种PRRSV毒株。新的抗病毒NPC(纳米抗体肽缀合物)由靶向病毒蛋白的特异性纳米抗体和用于病毒阻断的中和CD163表位肽组成,并提供显著的抗病毒活性。该研究将极大地促进针对PRRSV的抗病毒药物的研发,并启发针对其他病毒性疾病的新策略。
BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent swine pathogen, which has caused adverse impact on the global swine industry for almost 30 years. However, due to the immune suppression caused by the virus and the genetic diversity in PRRSV, no virus-targeting broad neutralizing strategy has been successfully developed yet. Antiviral peptide and nanobody have attracted extensive attention with the ease in production and the efficacy in practice. In this study, four new fusion proteins named nanobody peptide conjugates (NPCs) were developed by combining PRRSV specific non-neutralizing nanobodies with CD163-derived peptides targeting the receptor binding domain (RBD) of PRRSV proteins.
RESULTS: Four NPCs were successfully constructed using two nanobodies against PRRSV N and nsp9 individually, recombining with two antiviral peptides 4H7 or 8H2 from porcine CD163 respectively. All four NPCs demonstrated specific capability of binding to PRRSV and broad inhibitory effect against various lineages of PRRSV in a dose-dependent manner. NPCs interfere with the binding of the RBD of PRRSV proteins to CD163 in the PRRSV pre-attachment stage by CD163 epitope peptides in the assistance of Nb components. NPCs also suppress viral replication during the stage of post-attachment, and the inhibitory effects depend on the antiviral functions of Nb parts in NPCs, including the interference in long viral RNA synthesis, NF-κB and IFN-β activation. Moreover, an interaction was predicted between aa K31 and T32 sites of neutralizing domain 4H7 of NPC-N/nsp9-4H7 and the motif 171NLRLTG176 of PRRSV GP2a. The motif 28SSS30 of neutralizing domain 8H2 of NPC-N/nsp9-8H2 could also form hydrogens to bind with the motif 152NAFLP156 of PRRSV GP3. The study provides valuable insights into the structural characteristics and potential functional implications of the RBD of PRRSV proteins. Finally, as indicated in a mouse model, NPC intranasally inoculated in vivo for 12-24 h sustains the significant neutralizing activity against PRRSV. These findings inspire the potential of NPC as a preventive measure to reduce the transmission risk in the host population against respiratory infectious agents like PRRSV.
CONCLUSIONS: The aim of the current study was to develop a peptide based bioactive compound to neutralize various PRRSV strains. The new antiviral NPC (nanobody peptide conjugate) consists of a specific nanobody targeting the viral protein and a neutralizing CD163 epitope peptide for virus blocking and provides significant antiviral activity. The study will greatly promote the antiviral drug R&D against PRRSV and enlighten a new strategy against other viral diseases.