Production of a high purity, C-tagged hepatitis B surface antigen fusion protein VLP vaccine for malaria expressed in Pichia pastoris under cGMP conditions.

Virus-like particles (VLPs) induce strong humoral and cellular responses and have formed the basis of some currently licensed vaccines. Here, we present the method used for the production of R21, a VLP-based anti-sporozoite malaria vaccine, under current Clinical Good Manufacturing Practice regulations (cGMP). Previous preclinical studies in BALB/c mice showed that R21 produced almost complete protection against sporozoite challenge with transgenic Plasmodium berghei parasites. Here, we have modified the preclinical production process to enable the production of sufficient quantities of highly pure, clinical-grade material for use in human clinical trials. The R21 construct was re-engineered to include a C-tag to allow affinity-based separation from the major contaminant alcohol oxidase 1 (AOX 1, ~74 kDa). To our knowledge, this is the first use of C-tag technology to purify a VLP vaccine candidate for use in human clinical trials. The R21 vaccine has shown high-level efficacy in an African Phase IIb trial, and multiple clinical trials are underway to assess the safety and efficacy of the vaccine. Our findings support the future use of C-tag platform technologies to enable cGMP-compliant biomanufacturing of high purity yeast-expressed VLP-based vaccines for early phase clinical trials when clinical grade material is required in smaller quantities in a quick time frame.

The distribution of the radial and musculocutaneous nerves in the brachialis muscle.

Few have examined the distribution of the radial nerve branch to brachialis, generally believed to be motor, within the muscle. We examined the right brachialis muscles of six dissecting room cadavers and found that four received a supply from the radial nerve. The radial nerve branch(es) supplied the inferolateral region of the muscle and was overlapped proximodistally and mediolaterally by the intramuscular branches of the musculocutaneous nerve, which lay on a more superficial plane. The results have implications for the anterolateral approach to the humerus for orthopedic surgery. Anterior splitting of the muscle will almost certainly damage the most lateral branches of the musculocutaneous nerve.

Increased vascularisation enhances axonal regeneration within an acellular nerve conduit.

Despite major advances in microsurgical techniques, the functional results of periphery nerve repair remain largely unsatisfactory. Furthermore, if a defect exists such that a nerve graft is required, the results are generally worse than those following a primary repair. The autologous nerve graft, the current 'gold standard', has inherent disadvantages and there has been a long quest to find a suitable alternative. The role of vascularisation in nerve regeneration is poorly defined and the aim of this work was to define and quantify the effects of increased vascularisation on nerve regeneration. Rat sciatic nerve defects (1 cm) were bridged with a silicone chamber containing vascular endothelial growth factor (VEGF; 500 or 700 ng/ml) in a laminin-based gel (Matrigel) known to support axonal regeneration. Chambers were harvested between 5 and 180 days to follow the progression of neural and vascular elements. Following immunohistochemical staining, computerised image analysis demonstrated that VEGF significantly increased vascular, Schwann cell and axonal regeneration within the chamber up to 30 days post-insertion, and stimulated regeneration of up to 78% more myelinated axons at 180 days, compared to plain Matrigel control. Furthermore, the non-linear vascular dose-response to VEGF was clearly reflected in the Schwann cell and axonal staining intensity, supporting the highly significant relationship between vascularisation and Schwann cell staining seen within the chamber (P <0.001). Target-organ re-innervation at 180 days was similarly enhanced by VEGF in an identical dose-dependant manner. VEGF at 500 ng/ml increased recovery of gastrocnemius muscle weight by 17% and footpad innervation by 51% (P <0.05) compared to control, indicating the long-term functional benefits of VEGF.