Inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirus- and/or exosome-delivered the artificial microRNAs targeting sialoadhesin and CD163 receptors.

BACKGROUND: The current vaccines failed to provide substantial protection against porcine reproductive and respiratory syndrome (PRRS) and the new vaccine development faces great challenges. Sialoadhesin (Sn) and CD163 are the two key receptors for PRRS virus (PRRSV) infection of porcine alveolar macrophages (PAMs), but the artificial microRNA (amiRNA) strategy targeting two viral receptors has not been described. METHODS: The candidate miRNAs targeting Sn or CD163 receptor were predicted using a web-based miRNA design tool and validated by transfection of cells with each amiRNA expression vector plus the reporter vector. The amiRNA-expressing recombinant adenoviruses (rAds) were generated using AdEasy Adenoviral Vector System. The rAd transduction efficiencies for pig cells were measured by flow cytometry and fluorescent microscopy. The expression and exosome-mediated secretion of amiRNAs were detected by RT-PCR. The knock-down of Sn or CD163 receptor by rAd- and/or exosome-delivered amiRNA was detected by quantitative RT-PCR and flow cytometry. The additive anti-PRRSV effect between the two amiRNAs was detected by quantitative RT-PCR and viral titration. RESULTS: All 18 amiRNAs validated were effective against Sn or CD163 receptor mRNA expression. Two rAds expressing Sn- or CD163-targeted amiRNA were generated for further study. The maximal rAd transduction efficiency was 62% for PAMs at MOI 800 or 100% for PK-15 cells at MOI 100. The sequence-specific amiRNAs were expressed efficiently in and secreted from the rAd-transduced cells via exosomes. The expression of Sn and CD163 receptors was inhibited significantly by rAd transduction and/or amiRNA-containing exosome treatment at mRNA and protein levels. Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes. The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains. CONCLUSION: These results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains. Our findings provide new evidence supporting the hypothesis that exosomes can also serve as an efficient small RNA transfer vehicle for pig cells.

Blocking porcine sialoadhesin improves extracorporeal porcine liver xenoperfusion with human blood.

BACKGROUND: Patients in fulminant hepatic failure currently do not have a temporary means of support while awaiting liver transplantation. A potential therapeutic approach for such patients is the use of extracorporeal perfusion with porcine livers as a form of "liver dialysis". During a 72-h extracorporeal perfusion of porcine livers with human blood, porcine Kupffer cells bind to and phagocytose human red blood cells (hRBC) causing the hematocrit to decrease to 2.5% of the original value. Our laboratory has identified porcine sialoadhesin expressed on Kupffer cells as the lectin responsible for binding N-acetylneuraminic acid on the surface of the hRBC. We evaluated whether blocking porcine sialoadhesin prevents the recognition and subsequent destruction of hRBCs seen during extracorporeal porcine liver xenoperfusion. METHODS: Ex vivo studies were performed using wild type pig livers perfused with isolated hRBCs for 72-h in the presence of an anti-porcine sialoadhesin antibody or isotype control. RESULTS: The addition of an anti-porcine sialoadhesin antibody to an extracorporeal porcine liver xenoperfusion model reduces the loss of hRBC over a 72-h period. Sustained liver function was demonstrated throughout the perfusion. CONCLUSIONS: This study illustrates the role of sialoadhesin in mediating the destruction of hRBCs in an extracorporeal porcine liver xenoperfusion model.

Virus-Mimicking Polymer Nanoparticles Targeting CD169+ Macrophages as Long-Acting Nanocarriers for Combination Antiretrovirals.

Monosialodihexosylganglioside (GM3)-presenting lipid-coated polymer nanoparticles (NPs) that recapitulate the sequestration of human immunodeficiency virus-1 (HIV-1) particles in CD169+ virus-containing compartments (VCCs) of macrophages were developed as carriers for delivery and sustained release of a combination of two antiretrovirals (ARVs), rilpivirine (RPV) and cabotegravir (CAB). RPV and CAB were co-loaded into GM3-presenting lipid-coated polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA) NPs without loss in potency of the drugs. GM3-presenting PLA NPs demonstrated the most favorable release properties and achieved inhibition of HIV-1 infection of primary human macrophages for up to 35 days. Intracellular localization of GM3-presenting PLA NPs in VCCs correlated with retention of intracellular ARV concentrations and sustained inhibition of HIV-1 infection. This work elucidates the design criteria of lipid-coated polymer NPs to utilize CD169+ macrophages as cellular drug depots for eradicating the viral reservoir sites or to achieve long-acting prophylaxis against HIV-1 infection.

Anti-Siglec-1 antibodies block Ebola viral uptake and decrease cytoplasmic viral entry.

Several Ebola viruses cause outbreaks of lethal haemorrhagic fever in humans, but developing therapies tackle only Zaire Ebola virus. Dendritic cells (DCs) are targets of this infection in vivo. Here, we found that Ebola virus entry into activated DCs requires the sialic acid-binding Ig-like lectin 1 (Siglec-1/CD169), which recognizes sialylated gangliosides anchored to viral membranes. Blockage of the Siglec-1 receptor by anti-Siglec-1 monoclonal antibodies halted Ebola viral uptake and cytoplasmic entry, offering cross-protection against other ganglioside-containing viruses such as human immunodeficiency virus type 1.