Induction of Hepatitis E Virus Anti-ORF3 Antibodies from Systemic Administration of a Muscle-Specific Adeno-Associated Virus (AAV) Vector

Figures

• 

  Figure 1. Experimental overview. (A) The full-length 7.2 kb HEV genome encodes the replicase ORF1, and a 2.2 kb subgenomic HEV RNA encodes capsid ORF2 and the small phosphoprotein ORF3 [43]. (B) HEV exists in two forms: a non-enveloped virion (nHEV) found in faeces/intracellularly and a quasi-enveloped virion (eHEV) found in the blood stream and cell culture supernatant. Whether ORF3 is (partially) presented on the outside of eHEV particles remains unclear. (C) Production of AAV vectors was accomplished by triple-transfection of HEK-293 cells with a recombinant AAV vector plasmid encoding ORF3 or YFP (yellow fluorescent protein), an AAV helper plasmid encoding rep of AAV2 together with the synthetic capsid gene MYO3, as well as an adenoviral helper plasmid. This was followed by a harvest of the AAVs from the cell pellet and their purification by iodixanol density gradient centrifugation, buffer exchange to PBS, titration of AAV genome copies, and particle concentration. (D) Initial blood (60–80 μL) was collected from the facial vein before intravenous injection of 1 × 1011 or 1 × 1012 AAV vector particles encoding ORF3 or the YFP control (always diluted in 100 μL PBS) into four mice each.…

• 

  Figure 2. Muscle-specific expression of HEV-ORF3 in AAVMYO3-transduced mice. HEV ORF3 copy numbers were quantified via RT-qPCR in the quadriceps femoris muscle (green), liver (grey), and spleen (orange) of mice transduced with the indicated amount of vg ORF3 AAVs. Results represent the mean of n = 2 ± SD. ORF3 protein expression was analysed in respective mouse tissues via Western blotting. ORF3 expression was detected using a rabbit anti-ORF3 polyclonal Ab, as well as housekeeping genes GADPH or actin using respective mAbs. Palmitoylated ORF3 = ~15 kDa, GAPDH = ~36 kDa, actin = ~42 kDa.

• 

  Figure 3. Induction of anti-ORF3 antibodies correlates with ORF3 expression levels in mice. (A) Correlation of ORF3 copy numbers (data from Figure 2) and ORF3 protein expression in the muscle with anti-ORF3 antibodies in the serum of ORF3 AAV-transduced mice. ORF3 expression in the muscle was detected using the rabbit anti-ORF3 polyclonal Ab described above. To visualise the anti-ORF3 antibodies, HEK-293 cells transfected to express ORF3 were lysed and loaded onto a 12% SDS-PAGE. After separation and transfer onto a PVDF membrane, ectopic ORF3 was detected in the presence of anti-ORF3 antibodies in the mouse sera and visualised by incubation with anti-mouse HRP-conjugated Abs. As a loading control, actin was detected using an anti-actin mAb. (B) Dynamics of antibody induction in mice over 10 weeks. HEK-293 cells transfected to express ORF3 were lysed and loaded onto a 12% SDS-PAGE. After separation and transfer, the membrane was cut into individual pieces and incubated with sera from mouse #2 (1 × 1011 ORF3 AAV) or mouse #1 (1 × 1012 ORF3 AAV) harvested at the indicated different time points post-AAV injection, followed by a secondary anti-mouse antibody. As a loading control, actin was…

• 

  Figure 4. Inhibition of extracellular HEV particles by anti-ORF3 antibodies and sera from ORF3 AAV-transduced mice. (A) Extracellular HEV particles harvested from hepatoma S10-3 cells electroporated with in vitro transcribed HEV RNA were incubated with the indicated antibodies for 1 h prior to infection of S10-3 cells (anti-ORF2 and ORF3 pAb 1:200, anti-ORF2 mAb mix 1:50, anti-ORF3 mAb mix 1:50). HEV infection was normalised to untreated extracellular HEV particles (black bar). To assess the background of nHEV particles, supernatant from S10-3 cells electroporated with ΔORF3 virus was also titrated (grey bar). (B) Extracellular HEV particles were incubated with a 1:50 dilution of serum from mice harvested 10 weeks post-ORF3 AAV transduction and titered on S10-3 cells. HEV infection was normalised to extracellular HEV particles incubated with mouse serum transduced with YFP (dark grey bar). HEV infections were quantified by staining ORF2-positive cells 7 days post-infection when HEV replication in cultured cells peaks. Results represent the mean of n = 3 ± SD. Statistical analysis was performed using an unpaired, two-tailed t-test with *: p < 0.05.