Baculovirus Expression Vector System (BEVS) as a tunable platform for biologics production
If the ongoing global pandemic COVID-19 has taught us anything, it is the importance of an efficient biologics manufacturing platform that is cost-effective, reliable and has high product yield and quality. The baculovirus expression vector system (BEVS) has proven to be a promising platform to produce recombinant proteins, vaccines, virus-like particles (VLPs), and/or other biologics. However, it is believed that certain regions in the wild-type AcMNPV baculovirus genome are not essential for their in vitro replication or protein production. Thus, the expression of genes that are not required for progeny virus and protein production in cell culture could be ‘an additional burden’ resulting in the unnecessary depletion of cellular resources. Moreover, the co-production of baculovirus and recombinant protein products in the culture supernatant complicates the downstream purification processes. This work aims to use CRISPR-Cas9 to systematically study the essential and non-essential AcMNPV sequences active in the late and very late infection phases and subsequently remove the non-essential genes to yield a reduced genome recombinant baculovirus for efficient production of foreign proteins and/or progeny viruses. Finally, to reduce the burden on purification processes, disruption of reduced genome AcMNPV genes that are essential for virion production by using engineered Cas9 cells would prevent baculovirus contamination in the supernatant and eliminate the need for a trans-complementing cell line.
Enveloped viruses and virus-like particles
Influenza, coronaviruses, and even herpes viruses have been studied in the Aucoin Lab. These enveloped viruses are and continue to be a threat, if not just a nuissance. Enveloped viruses are described this way because of the lipid coat they inherit from the membranes of the host cells they infect. Typically, these viruses are released from the cell by budding through the outer membrane where they are coated or 'enveloped' by this lipid coat.
Virus-like particles (VLP) are particles that resemble virions; however, they typically do not contain any genetic material. In some cases, they may; however, the process of taking up DNA in a VLP would be a random event. This is what would differentiate a VLP from a defective virus - a virus that may not be able to replicate but that in all other ways resembles the wild-type infectious virion. Virus-like particles are often generated via the expression of a subset of structural genes that come together to form a particle resembling a virus without its genome. These particles, are of interest as potential vaccines because they combine the immunogenicity of inactivated or attenuated viruses with the safety of other recombinant vaccine products. The characterization of enveloped VLPs is complicated and complex.
Characterization and Purification of Enveloped VLPs in Insect Cells
The insect-cell baculovirus expression vector system (IC-BEVS) is an efficient platform for VLP production, owing to its high productivity, scalability, inherent safety, and partial post-translational modification capability. Moreover, IC-BEVS is capable of producing complex, multi-protein, enveloped VLPs (eVLP). Over the last three decades, a significant number of different VLP products have been successfully produced in IC-BEVS, however, few have progressed beyond laboratory scale. The major bottleneck in transitioning from lab scale to industrial scale and clinical use is the difficulty of downstream processing. In particular, the removal of co-produced recombinant baculovirus vectors in a scalable way has yet to be achieved. This is a significant problem for eVLPs especially, as they share many biophysical properties with the recombinant baculovirus. eVLPs are of interest in vaccine production because many viruses which negatively impact humans are enveloped (e.g., influenza, coronaviruses, ebolaviruses). In an effort to bridge the gap between lab scale and industrial successes in eVLP production, this work aims to investigate the separation of recombinant baculovirus and eVLPs by combining upstream modification to control the raw product with downstream separation techniques to purify the eVLPs.
COVID Immunity in a Campus Population at the Dawn of Vaccine Distribution
COVID-19 is not going away. Therefore, we, as a society, need to understand how we will function with it present, especially in places where people gather in large numbers or a required to live in close proximity, like campuses of Universities and Colleges. What is required is an immunity profile of the symptomatic and asymptomatic individuals in these locations. Only when this has been collected can we begin to understand and establish the factors that lead to the greatest risks of contracting and spreading the disease. Even though the University of Waterloo (UWaterloo) is conducting the majority of their undergraduate and graduate classes online, there are online components that require students to be in the city; many students, especially international students in their upper years, have returned to live in the city: both on and off campus even though they are studying online. Given the non-sustainability of online learning beyond the short term, students will likely return to campus for some version of in-person learning in the future while the risk of SARS-CoV-2 infection remains present. Not only does the University of Waterloo pride itself on its didactic practices, there is an in-person social network that facilitates and strengthens learning that can not be recreated on-line, despite the overwhelming number of social media and networking platforms and apps. Furthermore, despite having fewer students on-campus at this time, research and support staff, as well as maintenance and custodial staff, continue to work on campus and graduate students enrolled in experimental research programs are gradually being allowed to return to their thesis work. Therefore, the campus community will be constantly at risk for SARS-CoV-2 transmission, despite the use of mitigating measures. UWaterloo has implemented a COVID19 molecular testing center at Health Services to serve the campus community, which is acting as a sample collection center for students, faculty and staff. Given that we are an engineering lab able to efficiently produce proteins via transfection of mammalian cells, we are generating high-quality ELISA assays which we are then using in conjunction with Brian Dixon's Lab and St-Mary's Hospital, to quantify symptomatic and asymptomatic individuals; to understand the differential spread of the virus, in different sexes, blood groups and age groups exposed to similar risk levels in approximately the same environment; to combine the data from the above studies to develop profiles of COVID19 symptomatic and asymptomatic individuals on a university campus; and to compare antibody and memory T cell responses in COVID19 patients (in conjunction with McMaster University).
Genetic Manipulation of Microbial Communities via Bioaugmentation
Targeted genetic manipulation of microbial populations in situ is an emergent biotechnology that is being explored to eliminate antibiotic resistance in pathogens. This can be done through bioaugmentation, a technique that introduces an engineered strain of bacteria that spreads a plasmid (i.e. a circular piece of DNA) to other native bacteria in the environment. While bioaugmentation with engineered bacteria has demonstrated proof-of-principle results in lab-grown monocultures, there is uncertainty on how it will perform in natural environments. This problem is difficult to address because environments like water, soil, and the mammalian gut are composed of microbial communities that have complex ecological interactions. Furthermore, some environments have unique spatial features that can influence the structure and composition of their respective microbial communities. To make sound and ethical progress on bioaugmentation, standardized tools are needed to predict the outcomes of genetically manipulating natural microbial communities. However, these tools are lacking even for simple cocultures grown in the lab. The long-term goal is to apply bioaugmentation to more complex microbial ecosystems. Working towards this goal, two premises will be explored: 1) how ecological interactions between microbes influences horizontal gene transfer, and 2) how bioaugmentation can alter the stability of a microbial community. This will be explored through experimental and computational approaches. The work will result in microbial community design tools that predict bioaugmentation performance and the consequences of artificially altering microbial communities.