Evaluation of the contribution of the minor envelope complex glycoprotein 3 to the porcine reproductive and respiratory syndrome virus 1 neutralizing antibody response

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Porcine reproductive and respiratory syndrome viruses (PRRSV) cause significant economic losses to the global swine industry. Current live attenuated vaccines are unable to effectively control PRRSV infection and strain variability, driven by rapid mutation, complicates the development of improved vaccines. Identification of the targets of broadly neutralizing antibodies is crucial for guiding next-generation vaccine design, yet PRRSV glycoproteins bearing conserved neutralizing epitopes remain poorly defined. To address this, recombinant soluble forms of PRRSV-1 envelope glycoproteins, GP2, GP3, and GP4 were produced using mammalian cells. The screening of serum from hyperimmune pigs demonstrated GP3 as the most highly recognized of the three glycoproteins. Fluorescently tagged GP3-tetramers were utilized to isolate single B cells from a hyperimmune pig via flow cytometry. Immunoglobulin VH and VL regions were amplified by RT-PCR and sequenced. Fifteen unique heavy and light chain pairs were cloned and expressed as recombinant monoclonal antibodies (mAbs). mAbs were evaluated for specificity, cross-reactivity, and neutralizing capacity. Antigenic sites targeted by GP3-specifc mAbs were mapped to several conserved linear sequences, but mAbs did not exhibit virus neutralizing activity in vitro, suggesting these may serve as decoy epitopes. An immunogenicity study with recombinant GP3, delivered as a protein subunit or via an RNA vector, also demonstrated a lack of neutralizing antibody response despite high GP3-binding antibody titers. Collectively, these data suggest PRRSV-1 GP3 is not a neutralizing antibody target. However, the workflow established for isolating porcine mAbs could be utilized in future research to further dissect the neutralizing antibody response to PRRSV.

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