A Novel Cre Recombinase-Mediated In Vivo Minicircle DNA (CRIM) Vaccine Provides Partial Protection against Newcastle Disease Virus

Abstract

Minicircle DNA (mcDNA), which contains only the necessary components for eukaryotic expression and is thus smaller than traditional plasmids, has been designed for application in genetic manipulation. In this study, we constructed a novel plasmid containing both the Cre recombinase under the phosphoglycerate kinase (PGK) promoter and recombinant lox66 and lox71 sites located outside the cytomegalovirus (CMV) expression cassette. The strictly controlled synthesis of Cre recombinase in vivo maintained the complete form of the plasmid in vitro, whereas the in vivo production of Cre transformed the parental plasmid to mcDNA after transfection. The newly designed Cre recombinase-mediated in vivo mcDNA platform, named CRIM, significantly increased the nuclear entry of mcDNA, followed by increased production of mRNA and protein, using enhanced green fluorescent protein (EGFP) as a model. Similar results were also observed in chickens when the vaccine was delivered by the regulated-delayed-lysis Salmonella strain 11218, where significantly increased production of EGFP was observed in chicken livers. Then, we used the HN gene of genotype VII Newcastle disease virus as an antigen model to construct the traditional plasmid pYL43 and the novel mcDNA plasmid pYL47. After immunization, our CRIM vaccine provided significantly increased protection against challenge compared with that of the traditional plasmid, providing us with a novel mcDNA vaccine platform. IMPORTANCE Minicircle DNA (mcDNA) has been considered an attractive alternative to DNA vaccines; however, the relatively high cost and complicated process of purifying mcDNA dramatically restricts the application of mcDNA in the veterinary field. We designed a novel in vivo mcDNA platform in which the complete plasmid could spontaneously transform into mcDNA in vivo. In combination with the regulateddelayed-lysis Salmonella strain, the newly designed mcDNA vaccine provides us with an elegant platform for veterinary vaccine development.

In Vivo Production of HN Protein Increases the Protection Rates of a Minicircle DNA Vaccine against Genotype VII Newcastle Disease Virus

“…In our previous study [ 2 ], we designed an in vivo mcDNA platform for veterinary vaccine application, named CRIM. In this novel construction, the Cre-recombinase was driven by the strictly eukaryotic promoter Ppgk, which showed almost no activity in an in vitro condition.…”

“…However, there are still some disadvantages regarding to the use of the DNA vaccine in field, including the relatively lower immune response level after intramuscular or intradermal injection, with or without electroporation by a gene gun. To overcome this problem, we have designed a novel minicircle DNA (mcDNA) vaccine platform previously named Cre-recombinase mediated in vivo mcDNA (CRIM), in which the complete plasmid could transform into mcDNA in vivo after immunization, yielding an increased humoral immune response and better protection against a wild-type NDV challenge [ 2 ]. However, we are still not satisfied with our novel CRIM system regarding to its relatively lower and slow humoral immune response, even though it is still higher than the traditional plasmid DNA vaccine, and we are trying to improve our construction further.…”

“…In this study, we designed a novel CRIM vector based on regulated delayed lysis Salmonella which could synthesize HN protein under an in vivo regulated promoter P PagC , in addition to the previous mcDNA construction pYL47 [ 2 ], named pYL87. The novel designed CRIM vector could be used as an mcDNA vaccine as well as a protein subunit vaccine at the same time, which would provide us with an option to improve the immune response of the DNA vaccine.…”

Lox’d in translation: contradictions in the nomenclature surrounding common lox-site mutants and their implications in experiments

“…Of the 21 articles that unambiguously showed the correct loxsite annotations, 18/21 used the same lox orientation as the original paper by Albert et al [8], with the lox directionality reading from right to left [8,12,26,[30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. In contrast, five of the eight papers with mis-labelled lox sites portray them from left to right [17,[20][21][22]24].…”

Improving therapeutic potential of non-viral minimized DNA vectors

“…They have even been used for the endogenous production of biologics [ 33 ]. Their impact has also been felt in the areas of, among others, anti-viral treatments [ 34 ], vaccination [ 35 ], and regenerative medicine [ 36 , 37 ].…”