# Scalable Production of Recombinant Adeno-Associated Virus Vectors Expressing Soluble Viral Receptors for Broad-Spectrum Inhibition of Porcine Reproductive and Respiratory Syndrome Virus Type 2

## TL;DR

Scientists created a scalable method to produce virus-fighting proteins that can broadly inhibit a major swine virus.

## Contribution

A scalable insect cell-baculovirus system for producing rAAV vectors with dual soluble viral receptors against PRRSV.

## Key findings

- Optimized conditions achieved a 170-fold increase in rAAV vector titer compared to conventional methods.
- SVRs reduced viral titers of diverse PRRSV strains by ~4.3 log in vitro.
- The platform offers a durable solution to combat PRRSV with broad-spectrum antiviral activity.

## Abstract

We developed a scalable insect cell–baculovirus system to produce rAAV vectors expressing dual soluble viral receptors (Sn4D-Fc/SRCR59-Fc) against PRRSV. By systematically optimizing baculovirus co-infection ratio (0.5:1.0), initial cell density (2.0 × 106 cells/mL), fetal bovine serum concentration (2%), and temperature (30 °C), we achieved a scalable production of rAAV-SRCR59-Fc/Sn4D-Fc vectors with a titer of 5.4 ± 0.9 × 109 infectious viral particles (IVPs)/mL in a 2 L bioreactor, representing a 170-fold increase compared with conventional flask-based methods. In vitro tests showed these SVRs reduced viral titers of diverse PRRSV strains by ~4.3 log, demonstrating broad-spectrum antiviral activity. This platform offers a durable, scalable solution to combat PRRSV, addressing vaccine limitations for swine health management.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a major threat to the global swine industry, causing significant economic losses. To address this, we developed a scalable recombinant adeno-associated virus (rAAV)-based strategy for the delivery of soluble viral receptors (SVRs) to treat and potentially eliminate PRRSV infections. This strategy involves fusing the virus-binding domains of two key cellular receptors, sialoadhesin (Sn4D) and CD163 (SRCR5-9), with an Fc fragment. We then used an insect cell–baculovirus expression vector system to produce the rAAV-SRCR59-Fc/Sn4D-Fc vector. Through a series of optimizations, we determined the best conditions for rAAV production, including a baculovirus co-infection ratio of 0.5:1.0, an initial insect cell density of 2.0 × 106 cells/mL, a fetal bovine serum concentration of 2%, and a culture temperature of 30 °C. Under these optimized conditions, we achieved a high titer of rAAV-SRCR59-Fc/Sn4D-Fc in a 2 L bioreactor, reaching 5.4 ± 0.9 × 109 infectious viral particles (IVPs)/mL. Notably, in vitro neutralization assays using a Transwell co-culture system demonstrated a 4.3 log reduction in viral titers across genetically diverse PRRSV-2 strains, including VR2332, JXA1, JS07, and SH1705. Collectively, this study provides a robust platform for large-scale rAAV production and highlights the potential of SVR-based gene therapy to address the antigenic diversity of PRRSV-2.