Lung directed antibody gene transfer confers protection against SARS-CoV-2 infection.

BACKGROUND: The COVID-19 pandemic continues to be a worldwide threat and effective antiviral drugs and vaccines are being developed in a joint global effort. However, some elderly and immune-compromised populations are unable to raise an effective immune response against traditional vaccines. AIMS: We hypothesised that passive immunity engineered by the in vivo expression of anti-SARS-CoV-2 monoclonal antibodies (mAbs), an approach termed vectored-immunoprophylaxis (VIP), could offer sustained protection against COVID-19 in all populations irrespective of their immune status or age. METHODS: We developed three key reagents to evaluate VIP for SARS-CoV-2: (i) we engineered standard laboratory mice to express human ACE2 via rAAV9 in vivo gene transfer, to allow in vivo assessment of SARS-CoV-2 infection, (ii) to simplify in vivo challenge studies, we generated SARS-CoV-2 Spike protein pseudotyped lentiviral vectors as a simple mimic of authentic SARS-CoV-2 that could be used under standard laboratory containment conditions and (iii) we developed in vivo gene transfer vectors to express anti-SARS-CoV-2 mAbs. CONCLUSIONS: A single intranasal dose of rAAV9 or rSIV.F/HN vectors expressing anti-SARS-CoV-2 mAbs significantly reduced SARS-CoV-2 mimic infection in the lower respiratory tract of hACE2-expressing mice. If translated, the VIP approach could potentially offer a highly effective, long-term protection against COVID-19 for highly vulnerable populations; especially immune-deficient/senescent individuals, who fail to respond to conventional SARS-CoV-2 vaccines. The in vivo expression of multiple anti-SARS-CoV-2 mAbs could enhance protection and prevent rapid mutational escape.

Lung-targeting lentiviral vector for passive immunisation against influenza.

When recombinant simian immunodeficiency virus (SIV) is pseudotyped with the F and HN glycoproteins from murine respiratory Sendai virus (rSIV.F/HN), it provides efficient lung cell targeting and lifelong transgene expression in the murine airways. We have shown that a single dose of rSIV.F/HN can direct stable expression of neutralising antibody against influenza in the murine airways and systemic circulation, and protects mice against two different influenza strains in lethal challenge experiments. These data suggest that rSIV.F/HN could be used as a vector for passive immunisation against influenza and other respiratory pathogens.

Diabetes is associated with posttranslational modifications in plasminogen resulting in reduced plasmin generation and enzyme-specific activity.

Diabetes is associated with hypofibrinolysis by mechanisms that are only partially understood. We investigated the effects of in vivo plasminogen glycation on fibrinolysis, plasmin generation, protein proteolytic activity, and plasminogen-fibrin interactions. Plasma was collected from healthy controls and individuals with type 1 diabetes before and after improving glycemia. Plasma-purified plasmin(ogen) functional activity was evaluated by chromogenic, turbidimetric, and plasmin conversion assays, with surface plasmon resonance employed for fibrin-plasminogen interactions. Plasminogen posttranslational modifications were quantified by mass spectrometry and glycation sites located by peptide mapping. Diabetes was associated with impaired plasma fibrin network lysis, which partly normalized upon improving glycaemia. Purified plasmin(ogen) from diabetic subjects had impaired fibrinolytic activity compared with controls (723 ± 16 and 317 ± 4 s, respectively; P < .01), mainly related to decreased fibrin-dependent plasmin generation and reduced protease activity (Kcat/KM 2.57 ± 1.02 × 10⁻³ and 5.67 ± 0.98 × 10⁻³ M⁻¹s⁻¹, respectively; P < .05). Nε-fructosyl-lysine residue on plasminogen was increased in diabetes compared with controls (6.26 ± 3.43 and 1.82 ± 0.95%mol, respectively; P < .01) with preferential glycation of lysines 107 and 557, sites involved in fibrin binding and plasmin(ogen) cleavage, respectively. Glycation of plasminogen in diabetes directly affects fibrinolysis by decreasing plasmin generation and reducing protein-specific activity, changes that are reversible with modest improvement in glycemic control.

Expression of the NS3 protease of cytopathogenic bovine viral diarrhea virus results in the induction of apoptosis but does not block activation of the beta interferon promoter.

Bovine viral diarrhea virus (BVDV; genus Pestivirus) can exist as two biotypes, cytopathogenic (CP) and non-cytopathogenic (NCP). The CP form differs from NCP by the continual expression of free non-structural protein 3 (NS3). CP BVDV infection of cultured cells induces apoptosis, whereas NCP BVDV infection has been reported to block the induction of beta interferon (IFN-beta). To investigate the viral mechanisms underlying these effects, NS3 or NS2-3 proteins of NCP and CP BVDV biotypes, together with the cognate NS3 co-factor NS4A, were expressed in cells, and their effect on apoptosis and induction of IFN-beta was investigated. Expression of NS3/4A resulted in increased activity of caspase-9 and caspase-3, indicating induction of the intrinsic apoptosis pathway. Mutational analysis revealed that a protease-inactive NS3/4A was unable to induce apoptosis, suggesting that NS3 protease activity is required for initiation of apoptosis during CP BVDV infection. The ability of NS2-3 to modulate activation of the IFN-beta promoter was also investigated. These studies confirmed that, unlike the related hepatitis C virus and GB virus-B, BVDV proteases are unable to inhibit TLR3- and RIG-I-dependent activation of the IFN-beta promoter. These data suggest that BVDV NS3/4A is responsible for regulating the levels of cellular apoptosis and provide new insights regarding the viral elements associated with CP biotype pathogenesis.