Pre-clinical evaluation of a quadrivalent HCV VLP vaccine in pigs following microneedle delivery.

The introduction of directly acting antiviral agents (DAAs) has produced significant improvements in the ability to cure chronic hepatitis C infection. However, with over 2% of the world's population infected with HCV, complications arising from the development of cirrhosis of the liver, chronic hepatitis C infection remains the leading indication for liver transplantation. Several modelling studies have indicated that DAAs alone will not be sufficient to eliminate HCV, but if combined with an effective vaccine this regimen would provide a significant advance towards achieving this critical World Health Organisation goal. We have previously generated a genotype 1a, 1b, 2a, 3a HCV virus like particle (VLP) quadrivalent vaccine. The HCV VLPs contain the core and envelope proteins (E1 and E2) of HCV and the vaccine has been shown to produce broad humoral and T cell immune responses following vaccination of mice. In this report we further advanced this work by investigating vaccine responses in a large animal model. We demonstrate that intradermal microneedle vaccination of pigs with our quadrivalent HCV VLP based vaccine produces long-lived multi-genotype specific and neutralizing antibody (NAb) responses together with strong T cell and granzyme B responses and normal Th1 and Th2 cytokine responses. These responses were achieved without the addition of adjuvant. Our study demonstrates that our vaccine is able to produce broad immune responses in a large animal that, next to primates, is the closest animal model to humans. Our results are important as they show that the vaccine can produce robust immune responses in a large animal model before progressing the vaccine to human trials.

Shear-sensitive nanocapsule drug release for site-specific inhibition of occlusive thrombus formation.

Essentials Vessel stenosis due to large thrombus formation increases local shear 1-2 orders of magnitude. High shear at stenotic sites was exploited to trigger eptifibatide release from nanocapsules. Local delivery of eptifibatide prevented vessel occlusion without increased tail bleeding times. Local nanocapsule delivery of eptifibatide may be safer than systemic antiplatelet therapies. SUMMARY: Background Myocardial infarction and stroke remain the leading causes of mortality and morbidity. The major limitation of current antiplatelet therapy is that the effective concentrations are limited because of bleeding complications. Targeted delivery of antiplatelet drug to sites of thrombosis would overcome these limitations. Objectives Here, we have exploited a key biomechanical feature specific to thrombosis, i.e. significantly increased blood shear stress resulting from a reduction in the lumen of the vessel, to achieve site-directed delivery of the clinically used antiplatelet agent eptifibatide by using shear-sensitive phosphatidylcholine (PC)-based nanocapsules. Methods PC-based nanocapsules (2.8 × 1012 ) with high-dose encapsulated eptifibatide were introduced into microfluidic blood perfusion assays and into in vivo models of thrombosis and tail bleeding. Results Shear-triggered nanocapsule delivery of eptifibatide inhibited in vitro thrombus formation selectively under stenotic and high shear flow conditions above a shear rate of 1000 s-1 while leaving thrombus formation under physiologic shear rates unaffected. Thrombosis was effectively prevented in in vivo models of vessel wall damage. Importantly, mice infused with shear-sensitive antiplatelet nanocapsules did not show prolonged bleeding times. Conclusions Targeted delivery of eptifibatide by shear-sensitive nanocapsules offers site-specific antiplatelet potential, and may form a basis for developing more potent and safer antiplatelet drugs.

Identification of a homologue of CD59 in a cyclostome: implications for the evolutionary development of the complement system.

We have employed a COS cell expression cloning procedure to isolate a full length cDNA clone encoding a hagfish leukocyte-associated membrane protein (HLMP1). The protein, which is identified by a monoclonal antibody (JB3) generated in our laboratory, is present on the majority of hagfish leukocytes and is also expressed on erythrocytes. The cDNA clone contained an open reading frame encoding a 120 residue polypeptide which exhibits 33% amino acid sequence identity with the precursor protein of human CD59, a leukocyte-associated membrane protein which regulates the action of the complement membrane attack complex on homologous cells. CD59 belongs to a family of structurally related glycoproteins which includes the Ly-6 proteins expressed on mouse lymphocytes. In addition to significant overall sequence homology HLMP1 shows conservation of 8 key cysteine residues with members of the CD59/Ly-6 family. Comparison of the hagfish sequence with that of the mature human CD59 protein suggested a processed protein consisting of 74 amino acids associated with the cell membrane via a GPI anchor. The latter was confirmed by immuno-flow cytometry following treatment of transfected COS cells with phospholipase. Phylogenetic analysis and tissue distribution of this protein in the hagfish are consistent with HLMP1 being a homologue of CD59. A three-dimensional model of HLMP1, constructed using the NMR-determined structure for human CD59 as a template, indicated conservation of a core structure of five strands of beta-sheet and a short helix stabilised by four disulfide bonds. These findings, when taken together with our previous identification of C5a-like chemotactic activity in LPS-activated serum, provide indirect evidence for the existence of the terminal lytic complement pathway (C5 to C9) in these primitive vertebrates.