A CRISPR/Cas9 and Cre/Lox system-based express vaccine development strategy against re-emerging Pseudorabies virus

Abstract

Virus evolves rapidly to escape vaccine-induced immunity, posing a desperate demand for efficient vaccine development biotechnologies. Here we present an express vaccine development strategy based on CRISPR/Cas9 and Cre/Lox system against re-emerging Pseudorabies virus, which caused the recent devastating swine pseudorabies outbreak in China. By CRISPR/Cas9 system, the virulent genes of the newly isolated strain were simultaneously substituted by marker genes, which were subsequently excised using Cre/Lox system for vaccine safety concern. Notably, single cell FACS technology was applied to further promote virus purification efficiency. The combination of these state-of-art technologies greatly accelerated vaccine development. Finally, vaccination and challenge experiments proved this vaccine candidate's protective efficacy in pigs and the promise to control current pseudorabies outbreak. This is, to our knowledge, the first successful vaccine development based on gene edit technologies, demonstrating these technologies leap from laboratory to industry. It may pave the way for future express antiviral vaccine development.

Figure 1. Characterization and sequence analysis of the re-emerging PRV virulent strain.

(a–c) Amino acid sequence alignments of the main antigen genes gB, gC and gD among PRV HNX strain, PRV Bartha strain and the previous pandemic strain PRV Ea. (d) 24 days after vaccination with the commercial PRV vaccine, rectal temperatures of piglets challenged with PRV HNX, Ea strains or DMEM and (e) the neutralizing ability of antisera generated against PRV HNX and PRV Ea strain were measured (six piglets per group).

Figure 2. Flowchart of the novel express vaccine development strategy.

(a) Overview of the strategy for vaccine development. Firstly, PRV virulent gene gE and TK were simultaneously substituted by GFP and mCherry respectively using CRISPR/Cas9 system assisted homologous recombination. Next, single cell FACS was applied to accelerate recombinant virus purification. Subsequently, GFP and mCherry selection genes were excised by Cre/Lox system. (b) Construction of the recombinant DNA donors. mCherry and GFP genes were flanked with LoxP and LoxN sites respectively in same orientation. Transcription start site (TSS) of gE and TK were indicated by arrow.

Figure 3. Simultaneous double virulent gene recombination using CRISPR/Cas9 System.

(a) Procedure of CRISPR/Cas9 system assisted double-gene recombination. (b) HEK293T cells co-transfected with sgRNAs and DNA donors and then infected with PRV HNX (MOI = 1). Upon recombination, GFP and mCherry expression will be driven by viral TK and gE promoter respectively. (c) Single cells with fluorescent gene recombinant virus were sorted by FACS, plated to 96 well plate pre-cultured with PK15 cells and incubated until fluorescence appeared. (d) Plaque purification of double genes recombinant virus in agarose-DMEM plates. Cell plaques were indicated by arrows. (e) PCR verification of TK and gE gene deletion.

Figure 4. Double fluorescent gene excision using Cre/Lox System.

(a) Procedure of double fluorescent gene excision by Cre/Lox system. HEK293T cells were transfected with Cre-recombinase (b) or control plasmids (c) and then infected with fluorescent gene recombinant virus (MOI = 1). The images were taken at 36 hours after infection. (d) Plaque purification for double fluorescent genes excision virus. Cell plaques were indicated by arrows.

Figure 5. Protective effect of PRV-HNX TK⁻/gE⁻ in mice.

(a) Two group of mice were subcutaneously injected with PRV HNX or PRV-HNX TK⁻/gE⁻ and were monitored for survival rate for two weeks. Mice were vaccinated PRV-HNX TK⁻/gE⁻ or DMEM and then challenged with PRV HNX (b), PRV Ea (c) and PRV-Becker-GFP (d) two weeks later. Survival rate were monitored for two weeks. (e) Fluorescence imaging of the brain stem section of the mice vaccinated PRV-HNX TK⁻/gE⁻ or DMEM respectively and then challenged with PRV-Becker-GFP (five mice per group).

Figure 6. Protective effect of PRV-HNX TK⁻/gE⁻ in piglets.

Two groups of piglets were vaccinated with PRV-HNX TK⁻/gE⁻ or DMEM respectively, and then challenged with PRV HNX. (a) Survival rate, (b) gB-specific antibody ELISA, (c) body temperature, and (d) average daily weight gain were recorded. (e,f) Histological imaging of the brain section of the piglets vaccinated PRV-HNX TK⁻/gE⁻ or DMEM respectively, and then challenged with PRV HNX. (≤0.6 = positive, 0.6–0.7 = suspect, >0.7 = negative).