Stability of plasmid and viral banks supporting the cGMP manufacture of Q-Griffithsin from a TMV-based viral vector.
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ABSTRACT:
SUBMITTER: Corman JM
PROVIDER: S-EPMC7397727 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:To prevent sexually transmitted HIV, the most desirable active ingredients of microbicides are antiretrovirals (ARVs) that directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals, which are costly to manufacture and deliver to resource-poor areas where effective microbicides are urgently needed. Here, we report a manufacturing breakthrough for griffithsin (GRFT), one of the most potent HIV entry inhibitors. This red algal protein was produced in multigram quantities after extraction from Nicotiana benthamiana plants transduced with a tobacco mosaic virus vector expressing GRFT. Plant-produced GRFT (GRFT-P) was shown as active against HIV at picomolar concentrations, directly virucidal via binding to HIV envelope glycoproteins, and capable of blocking cell-to-cell HIV transmission. GRFT-P has broad-spectrum activity against HIV clades A, B, and C, with utility as a microbicide component for HIV prevention in established epidemics in sub-Saharan Africa, South Asia, China, and the industrialized West. Cognizant of the imperative that microbicides not induce epithelial damage or inflammatory responses, we also show that GRFT-P is nonirritating and noninflammatory in human cervical explants and in vivo in the rabbit vaginal irritation model. Moreover, GRFT-P is potently active in preventing infection of cervical explants by HIV-1 and has no mitogenic activity on cultured human lymphocytes.
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Project description:Griffithsin, a broad-spectrum antiviral lectin, has potential to prevent and treat numerous viruses including HIV, HCV, HSV, SARS-CoV, and SARS-CoV-2. For these indications, the annual demand for Griffithsin could reach billions of doses and affordability is paramount. We report the lab-scale validation of a bioprocess that supports production volumes of >20 tons per year at a cost of goods sold below $3,500/kg. Recombinant expression in engineered E. coli enables Griffithsin titers ∼2.5 g/L. A single rapid precipitation step provides > 90% yield with 2-, 3-, and 4-log reductions in host cell proteins, endotoxin, and nucleic acids, respectively. Two polishing chromatography steps remove residual contaminants leading to pure, active Griffithsin. Compared to a conventional one this process shows lower costs and improved economies of scale. These results support the potential of biologics in very large-scale, cost-sensitive applications such as antivirals, and highlight the importance of bioprocess innovations in enabling these applications.
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Project description:Intramuscular injection of an Adeno-associated viral vector serotype 1 (AAV1) encoding Neurotrophin-3 (NT3) into hindlimb muscles 24 h after a severe T9 spinal level contusion in rats has been shown to induce lumbar spinal neuroplasticity, partially restore locomotive function and reduce spasms during swimming. Here we investigate whether a targeted delivery of NT3 to lumbar and thoracic motor neurons 48 h following a severe contusive injury aids locomotive recovery in rats. AAV1-NT3 was injected bilaterally into the tibialis anterior, gastrocnemius and rectus abdominus muscles 48-h following trauma, persistently elevating serum levels of the neurotrophin. NT3 modestly improved trunk stability, accuracy of stepping during skilled locomotion, and alternation of the hindlimbs during swimming, but it had no effect on gross locomotor function in the open field. The number of vGlut1+ boutons, likely arising from proprioceptive afferents, on gastrocnemius α-motor neurons was increased after injury but normalised following NT3 treatment, suggestive of a mechanism in which functional benefits may be mediated through proprioceptive feedback. Ex vivo MRI revealed substantial loss of grey and white matter at the lesion epicentre but no effect of delayed NT3 treatment to induce neuroprotection. Lower body spasms and hyperreflexia of an intrinsic paw muscle were not reliably induced in this severe injury model suggesting a more complex anatomical or physiological cause to their induction. We have shown that delayed intramuscular AAV-NT3 treatment can promote recovery in skilled stepping and coordinated swimming, supporting a role for NT3 as a therapeutic strategy for spinal injuries potentially through modulation of somatosensory feedback.
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