A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

Abstract

A “universal” vaccine design platform that can rapidly generate multiplex vaccine candidates is critically needed to control future pandemics. Here, using SARS-CoV-2 pandemic virus as a model, we have developed such a platform by CRISPR engineering of bacteriophage T4. A pipeline of vaccine candidates were engineered by incorporating various viral components into appropriate compartments of phage nanoparticle structure. These include: expressible spike genes in genome, spike and envelope epitopes as surface decorations, and nucleocapsid proteins in packaged core. Phage decorated with spike trimers is found to be the most potent vaccine candidate in mouse and rabbit models. Without any adjuvant, this vaccine stimulated robust immune responses, both TH1 and TH2 IgG subclasses, blocked virus-receptor interactions, neutralized viral infection, and conferred complete protection against viral challenge. This new type of nanovaccine design framework might allow rapid deployment of effective phage-based vaccines against any emerging pathogen in the future.

CRISPR Engineering of Bacteriophage T4 to Design Vaccines Against SARS-CoV-2 and Emerging Pathogens

“…The resulting pET-28b-Soc-RBD donor plasmid (kanamycin-resistant) was confirmed by sequencing using Soc-BglII-F and T7term primers. Plaque assays were performed to determine the efficiency of the Cas12a-Hoc or Soc spacer to restrict T4 phage infection [19,32].…”

“…9. PCR analysis was performed to confirm Ee or RBD gene insertion [19]. One microliter of G3 phages was denatured at 94 C for 8 min and used as a template for PCR using Master Mix and specific primers (see Note 16).…”

“…All these would necessitate the installation of second-generation vaccines that can provide more robust, lasting, and broader protection. With these in mind, we have recently developed a "universal" bacteriophage (phage) T4 vaccine delivery platform for designing COVID-19 vaccines using CRISPR genome editing technology [19].…”

“…We recently reported the development of CRISPR engineering technology [32,33] by which foreign genes can be inserted into phage genome in an expressible form [19] (Fig. 1).…”

“…Taking advantage of the unique genetic and structural features of bacteriophage T4 and combining these with CRISPR phage engineering, we proposed a powerful "bacteriophage as vaccine" approach to rapidly design coronavirus vaccines [19]. Furthermore, the T4 vaccines do not require an adjuvant and can be produced by growing the recombinant phage in E. coli, allowing rapid construction and relatively inexpensive manufacturing and global distribution of the vaccine.…”