Killed whole genome-reduced bacteria surface-expressed coronavirus fusion peptide vaccines protect against disease in a porcine model

Abstract

As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible Gram-negative autotransporter to express vaccine antigens on surface of genome-reduced bacteria to enhance interaction of vaccine antigen with immune system. As a proof of principle, we utilized genome-reduced E. coli to express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as a killed whole cell vaccine. The FP sequence is highly conserved across coronaviruses; the 6 FP core amino acid residues along with the 4 adjacent residues upstream and the 3 residues downstream the core are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated IFN-γ responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly-protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens.

Minicells from Highly Genome Reduced Escherichia coli: Cytoplasmic and Surface Expression of Recombinant Proteins and Incorporation in the Minicells

“…We then cured the strain of kanamycin resistance by recombination with a derivative of pCP20 22, 23 and verified the removal of the kanamycin resistance gene phenotypically, confirmed removal with sequencing, and reconfirmed the minicell production phenotype as described below. To examine expression in the minicells made from the genome reduced bacteria, we used a plasmid, pMP2463 that expresses GFP, and we commissioned the synthesis of a plasmid, pRAIDA2 (FIG 1), that includes an AIDA-I-based autotransporter expression cassette 24, 25 . In its parental form, pRAIDA2 has an influenza HA immunotag in the autotransporter expression cassette.…”

“…To show that minicells made from genome reduced bacteria can contain recombinant proteins within their cytoplasm, we transformed the minCminD mutants of the wild type (ME5000) and the most highly genome reduced strain of E. coli in the TMUG collection (ME5125) 4, 5 with a GFP expressing plasmid, pMP2463 26 . We also transformed the undeleted ME 5000 and 29.7% deleted ME 5125 stains with minCminD mutations with pRAIDA2, the plasmid that expresses an HA immunotag via an AIDA-I autotransporter surface expression cassette under the control of a rhamnose-inducible promoter 25 .…”

“…We recently showed that killed whole cell E. coli vaccines that expressed antigens derived from the fusion peptides of either SARS-CoV-2 or porcine epidemic diarrhea virus (PEDV) can elicit good anamnestic responses and protect against disease in pigs in an experimental PEDV infection model. However, this approach did not elicit a strong neutralizing antibody response 25 ; an analogous minicell vaccine with an enhanced amount of antigen may yield improved immune responses. Approved killed whole cell vaccines have been produced at a cost of <1 US$ per dose 28 , and we suggest that at-scale production of minicell vaccines should be not be substantially higher.…”

“…We commissioned the synthesis of pRAIDA2 (FIG 1) (Genesys). pRAIDA2 includes an expression cassette based on the AIDA-I 24 autotransporter 17, 32 under the control of a rhamnose-inducible promoter, an origin of replication, and a kanamycin resistance gene 25 . The parental version of pRAIDA2 expresses an influenza HA immunotag on a stuffer fragment within the expression cassette’s cloning site.…”

A human cell-based SARS-CoV-2 vaccine elicits potent neutralizing antibody responses and protects mice from SARS-CoV-2 challenge

“…As the oldest member of vaccine family with a history of success, inactivated vaccines remain a major type of vaccines employed against many pathogens including influenza and SARS-CoV-2. For making inactivated vaccines, virus or bacteria was propagated in culture and then exposed to chemical or physical agents to destroy its replicative activity while maintain a normal display of its surface proteins and thus leave the immunogenicity largely intact [13]. Inspired by the working mechanism of inactivated vaccine, we herein added to the roadmaps of SARS-CoV-2 vaccine a new avenue, that is, using cultured human cells as carrier to express S protein in its native membrane-bound form with their safety as vaccines being secured by inactivation without loss of immunogenicity.…”