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The insect larvae/baculovirus protein production process was dramatically simplified by expressing fusion proteins containing green fluorescent protein (GFP) and the product-of-interest. In this case, human interleukin-2 (hIL-2) and chloramphenicol acetyl-transferase (CAT) were model products. Specifically, our fusion construct was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), the UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of the product from the fusion, and the product, hIL-2 or CAT. Both the approximately 52 kDa GFPuv/hIL-2 and approximately 63 kDa GFPuv/CAT fusions were expressed in Trichoplusia ni larvae at 9.0 microg-hIL-2 and 24.1 microg-CAT per larva, respectively. The GFP enabled clear identification of the infection process, harvest time, and more importantly, the quantity of product protein. Because the GFP served as a marker, this technique obviates the need for in-process Western analyses (during expression, separation, and purification stages). As a purification marker, GFP facilitated rapid identification of product-containing elution fractions (Cha et al., 1999b), as well as product-containing waste fractions (e.g., cell pellet). Also, because the fluorescence intensity was linear with hIL-2 and CAT, we were able to select the highest-producing larvae. That is, three fold more product was found in the brightest larva compared to the average. Finally, because the GFP is attached to the product protein and the producing larvae can be selected, the infection and production processes can be made semi-continuous or continuous, replacing the current batch process. These advantages should help to enable commercialization of larvae as expression hosts.

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Viral hemorrhagic septicemia virus (VHSV), a member of the Novirhabdovirus genus, contains an 11-nucleotide conserved sequence at the terminal 3'- and 5'-untranslated regions (UTRs) that are complementary. To study the importance of nucleotides in the 3'-UTR of VHSV for replication of novirhabdoviruses, we performed site-directed mutagenesis of selected residues at the 3'-terminus and generated mutant viruses using a reverse genetics approach. Assessment of growth kinetics and in vitro real-time cytopathogenicity studies showed that the order of two nucleotides (A4G5) of the 3'-terminus of VHSV directly affects growth kinetics in vitro. The mutant A4G-G5A virus has reduced total positive-strand RNA synthesis efficiency (51% of wild-type) at 48h post-transfection and 70h delay in causing complete cytopathic effect in susceptible fish cells, as compared to the WT-VHSV. Furthermore, when the A4G-G5A virus was used to challenge zebrafish, it exhibited reduced pathogenicity (54% lower end-point mortality) compared to the WT-VHSV. From these studies, we infer that specific residues in the 3'-UTR of VHSV have a promoter function and are essential to modulate the virulence in cells and pathogenicity in fish.

The COVID-19 pandemic has claimed the lives of millions of people worldwide and threatens to become an endemic problem, therefore the need for as many types of vaccines as possible is of high importance. Because of the millions of doses required, it is desirable that vaccines are not only safe and effective, but also easy to administer, store, and inexpensive to produce. Newcastle Disease Virus (NDV) is responsible for a respiratory disease in chickens. It has no pathogenic homologue in humans. NDV is recognized as an oncolytic virus, and its use in humans for oncological treatment is being evaluated. In the present work, we have developed two types of NDV-vectored candidate vaccines, which carry the surface-exposed RBD and S1 antigens of SARS-CoV-2, respectively. These vaccine candidates were produced in specific-pathogen-free embryonating chicken eggs, and purified from allantoic fluid before lyophilization. These vaccines were administered intranasally to three different animal models: mice, rats and hamsters, and evaluated for safety, toxicity, immunogenicity, stability and efficacy. Efficacy was evaluated in a challenge assay against active SARS-CoV-2 virus in the Golden Syrian hamster model. The NDV-vectored vaccine based on the S1 antigen was shown to be safe and highly immunogenic, with the ability to neutralize SARS-CoV-2 in-vitro, even with an extreme dilution of 1/640. Our results reveal that this vaccine candidate protects the lungs of the animals, preventing cellular damage in this tissue. In addition, this vaccine reduces the viral load in the lungs, suggesting that it may significantly reduce the likelihood of transmission. Being lyophilized, this vaccine candidate is very stable and can be stored for several months at 4-8°C. In conclusion, our NDV-based vaccine candidate has shown a very favorable performance in the pre-clinical study, serving as evidence for a future evaluation in a Phase-I human clinical trial. This candidate represents a promising tool in the fight against COVID-19.

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