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

Abstract

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.

Keywords: PRRSV; adeno-associated virus vector; broad-spectrum antiviral activity; insect cell bioreactor; soluble viral receptor fusions.

Figure 1

Optimization of rAAV production parameters. (A) Effect of baculovirus co-infection ratio (rBac-SRCR59-Fc/Sn4D-Fc:rBac-RC) on rAAV packaging efficiency. Sf9 cells (1.0 × 10⁶ cells/mL) were co-infected at MOI ratios of 0.1:1.0, 0.5:1.0, 1.0:1.0, 5.0:1.0, or 10:1.0. The highest rAAV titer (4.0 ± 0.5 × 10⁶ IVPs/mL) was achieved at an MOI ratio of 0.5:1.0, which was significantly higher than other ratios (p < 0.01 for 5.0:1.0 and 10:1.0; p < 0.05 for 1.0:1.0 and 0.1:1.0). (B) Impact of initial insect cell density on rAAV yield. Maximum production (6.1 ± 0.1 × 10⁶ IVPs/mL) occurred at 2.0 × 10⁶ cells/mL, showing significant improvement compared to 0.5 × 10⁶ (p < 0.01), 3.0 × 10⁶ (p < 0.01), and 1.0 × 10⁶ (p < 0.05) cells/mL. (C) Influence of fetal bovine serum (FBS) concentration on rAAV production. Optimal yields were observed in 2% FBS medium (6.1 ± 0.1 × 10⁶ IVPs/mL), which was significantly higher than serum-free conditions (p < 0.01) but showed no significant difference from 5% or 10% FBS groups. (D) Effect of culture temperature on rAAV titers. The highest titer (1.9 ± 0.3 × 10⁷ IVPs/mL) was achieved at 30 °C, significantly surpassing 27 °C (p < 0.01) and 24 °C (p < 0.01). No infectious virus was detected at 33 °C. No infectious virus was detected at 33 °C. ** indicates a highly significant difference (p < 0.01) and * indicates a significant difference (p < 0.05) between experimental groups (two-tailed Student’s t-test). Data are presented as mean ± SD from three independent biological replicates. Error bars represent SD.

Figure 2

Time course of baculovirus co-infection in the cell flask. To determine the optimum harvest time of BIICs under the optimized baculovirus co-infection conditions, the insect cell density, the number of living cells, and the yields of rAAV were checked at different times after infection.

Figure 3

High yields of recombinant adeno-associated virus packaging. (A) The protocol of the rAAV-SRCR59-Fc/Sn4D-Fc large-scale production method. (B) Time course of rAAV production in the insect cell bioreactor. Sf9 cells (7.5 × 10⁵/mL) were grown in the 2 L bioreactor. Specifically, the cells were fed-batch with fresh medium twice, 48 h and 24 h before infection. BIICs were added to reach the final working volume when the cell density reached 2 × 10⁶ cells/mL (t = 0 hpi). Subsequently, the total insect cell density, viable insect cell density, and rAAV titer were measured every 24 h until 192 h post-infection.

Figure 4

Identification of rAAV particles and SVR expression in rAAV-transduced 3D4/21 cells. (A) Purified rAAV-SRCR59-Fc/Sn4D-Fc was stained with 3% phosphotungstic acid and observed under a transmission electron microscope at an acceleration voltage of 75 kV. (B) The structural proteins of rAAV were detected by Western blotting using anti-AAV serum as the primary antibody. (C) 3D4/21 cells were transduced with rAAV-SRCR59-Fc/Sn4D-Fc and analyzed by immunofluorescence using anti-pig IgG. (D) The SVR fusions were purified from the cell medium of rAAV-transduced cells using Protein A affinity columns and analyzed by Western blotting using anti-Sn or anti-CD163 serum. D/I ratios represent target protein densitometry values normalized to loading control using Image J software 1.8.0. Western blot original images can be found in Supplementary Materials.

Figure 5

Antiviral activity of rAAV-SRCR59-Fc/Sn4D-Fc against diverse PRRSV-2 strains. 3D4/21 cells were transduced with rAAV-SRCR59-Fc/Sn4D-Fc (MOI 100) or empty AAV control. PAM cells were infected with PRRSV strains (MOI 0.5) and co-cultivated with transduced 3D4/21 cells for 24 h. Viral titers in PAM lysates were determined by TCID₅₀ assay on Marc-145 cells. ** indicates highly significant difference (p < 0.01) between the empty AAV control and rAAV-SRCR59-Fc/Sn4D-Fc transduction experimental group. Error bars represent SD (n = 3).