Manufacturing has been the key factor limiting rollout of vaccination during the COVID-19 pandemic, requiring rapid development and large-scale implementation of novel manufacturing technologies. ChAdOx1 nCoV-19 (AZD1222, Vaxzevria) is an efficacious vaccine against SARS-CoV-2, based upon an adenovirus vector. We describe the development of a process for the production of this vaccine and others based upon the same platform, including novel features to facilitate very large-scale production. We discuss the process economics and the "distributed manufacturing"

In anticipation of large-scale smallpox vaccination, clinical trials of new vaccine candidates with improved safety profiles, and new vaccinia immune globulin (VIG) products, there is an immediate need to develop new assays to measure vaccinia-specific immune responses. The classical assay to measure vaccinia neutralization, the plaque-reduction neutralization test (PRNT), is slow, labor intensive, and difficult to validate and transfer. Here we describe the development of a novel vaccinia-neutralization assay based on the expression of a reporter gene, beta-galactosidase (beta-Gal). Using a previously constructed vaccinia-beta-Gal recombinant virus, vSC56, we developed a neutralization assay that is rapid, sensitive, and reproducible. The readout is automated. We show that the neutralizing titers, ID(50), for several VIG products measured by our assay were similar to those obtained by PRNTs. A new Food and Drug Administration VIG standard was established for distribution to other laboratories. The new assay will serve as an important tool both for preclinical and clinical trials of new smallpox vaccines and for evaluation of therapeutic agents to treat vaccine-associated adverse reactions.

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A recombinant vaccinia virus was engineered to express enhanced green fluorescent protein (EGFP) under control of the T7 promoter using the VOTE expression system in HeLa cells. Infection of HeLa cells with this virus and induction with IPTG demonstrated the utility of this construct for easily measuring protein expression. This construct was used to evaluate several production parameters, specifically, multiplicity of infection (MOI), volume during infection, and serum concentration during the infection phase. In static culture, increasing multiplicity of infection was found to increase expression of EGFP up to a plateau around MOI of 1.0. Expression was also shown to increase with decreasing volume during the infection phase. Serum concentration during the infection phase was only marginally significant from 0 to 7.5%. Cytodex 3 microcarriers were found to have the best characteristics for HeLa cell growth. These cells were grown and infected in microcarrier spinner flask culture, and the maximum expression was 2.2 microg EGFP/(million cells at the time of infection), demonstrating the ability of this system to successfully express recombinant proteins at larger scale.