Conceived and designed the experiments: NJ XL ZD. Performed the experiments: SK YW LS PJ JS LL LL GY ZW. Analyzed the data: SK YW LS PJ. Contributed reagents/materials/analysis tools: YQ JW GL. Wrote the paper: SK.
The authors have declared that no competing interests exist.
Vaccinia Tian Tan (VTT) was attenuated by deletion of the TC7L-TK2L and TA35R genes to generate MVTT3. The mutant was generated by replacing the open reading frames by a gene encoding enhanced green fluorescent protein (EGFP) flanked by loxP sites. Viruses expressing EGFP were then screened for and purified by serial plaque formation. In a second step the marker EGFP gene was removed by transfecting cells with a plasmid encoding cre recombinase and selecting for viruses that had lost the EGFP phenotype. The MVTT3 mutant was shown to be avirulent and immunogenic. These results support the conclusion that TC7L-TK2L and TA35R deletion mutants can be used as safe viral vectors or as platform for vaccines.
Vaccinia virus Tian Tan (VTT) strain has been studied for use as a live viral vector for infectious diseases or cancer therapy
Researchers are increasingly utilizing conditional gene manipulation strategies that allow deletion of a gene of interest
In the present study, we constructed a modified VTT genome (MVTT3) by deleting both TC7L-TK2L and TA35R genes, which resulted in reduced virulence. Deletion of small DNA fragments (3–25 nucleotides) are common in poxviruses
BHK-21 hamster kidney cells, PK(15) porcine kidney cells, HeLa human cervical adenocarcinoma epithelial cells, Madin-Darby canine kidney (MDCK) epithelial cells, and Vero African green monkey kidney cells were obtained from the China Center for Type Culture Collection. The BHK-21 and PK(15) cells were cultured in modified Eagle's medium (MEM). HeLa and MDCK cells were cultured in Dulbecco's MEM. The Vero cells were cultured in RPMI 1640. Culture media (Invitrogen, Beijing, China) for the HeLa, MDCK, and Vero cells were supplemented with 10% fetal bovine serum (FBS; Hyclone, Beijing, China), 100 units/ml benzyl penicillin, and 100 µg/ml streptomycin sulfate. Media for the BHK-21 and PK(15) cells were supplemented with 5% FBS, 100 units/ml benzyl penicillin, and 100 µg/ml streptomycin sulfate. The parental vaccinia Tian Tan (GenBank accession no. AF095689) strain was obtained from the Institute of Virology at the Chinese Center for Disease Control and Prevention. Three- to five-week-old female BALB/c mice and female New Zealand white rabbits were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences of China. The animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Chinese Academy of Military Medical Science, Changchun, China (10ZDGG007). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
The plasmid pVAX1-Cre, a derivative of pVAX1, was provided by Xiaoming Xia. Standard gene synthesis techniques were used to construct shuttle plasmid pSK-PTC7L-TK2L-EGFP and pSK-PTA35R-EGFP. A DNA fragment containing TC7L–loxP–PE/L–EGFP–loxP–TK2L sites was constructed by Shanghai Generay Biotech Co., Ltd. The PE/L–EGFP cassette was flanked by similarly oriented loxP sites. Replacement of the TC7L-TK2L gene by EGFP was confirmed using TC7LSP (
Homologous recombination resulting in TC7L-TK2L replaced by the EGFP open reading frame (ORF) generated recombinant MVTT1-EGFP. This was accomplished by infecting 80% confluent BHK-21 cells with an input multiplicity of infection (MOI) of 0.01 PFU VTT per cell
BHK-21 cells (1×104) were infected with 100 PFU MVTT1-EGFP. At 2 h post-infection, the cells were transfected with shuttle plasmid pVAX1-Cre. Forty-eight hours after co-transfection, non-EGFP-expressing plaques were picked and isolated by five rounds of plaque purification. The MVTT1 was propagated in BHK-21 cells. PCR amplification was used to confirm the deletion of EGFP. The MVTT1 genomic DNA was extracted as template, the primers TC7LSP1 and TK2LASP1 through 30 cycles at 95°C for 5 min, 94°C for 30 s, 55°C for 45 s, and 72°C for 45 s with the last extension at 72°C for 10 min. The product was resolved by 1% agarose gel electrophoresis.
Recombinant MVTT2 was constructed using the same strategy described above. The TA35R gene was knocked out successively by homologous recombination with pSK-PTA35R-EGFP, and removal of EGFP by the Cre-loxP system. The double deletions were confirmed using the same PCR amplifications as shown above.
The MVTT1 strain was used in a second homologous recombination. A similar procedure was used to delete the TA35R ORF from the MVTT1 strain to form MVTT3. The double deletions were confirmed using the same PCR amplification as it was shown above.
Monslayers of BHK-21 cells were infected with the three mutants at 100 PFU and serially passaged 10 times to evaluate their genetic stability
The test used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, St. Louis, MO). The BHK-21, MDCK, HeLa, Vero, and PK(15) cells were seeded in 96-well plates (1×104 cells/well) 1 d before they were infected with various concentrations (0.05 MOI) of MVTT1, MVTT2, MVTT3, or VTT. The viability of the five cell lines were tested every 24 h by treating cells with 20 µl of MTT (5 mg/ml) and incubating for 4 h at 37°C. The culture medium was removed, and the formed crystals were dissolved by exposing the cells in each well to 150 µl dimethylsulfoxide for 10 min at 37°C. The absorbance of the suspension at 490 nm was measured by an enzyme-linked immunosorbent assay (ELISA) plate reader. Untreated cells were used as controls, and all samples were examined thrice. The cell survival rate was calculated using the following formula: 100×(Experiment absorbances)/(control absorbances)
To test the spread efficiently of mutants in different cells, plaques of the mutants were visualized by crystal violet staining. The three mutants MVTT1, MVTT2 and MVTT3 at 0.05 PFU/cell were added to the five cell lines in 12-well plates. The cells were overlaid with 1% agarose after 90 min of viral exposure and then incubated for another 48 h at 37°C. Cellular growth was stopped by removing the medium, and 0.5% crystal violet in 20% methanol was added to the cells for 10 minutes. CPE was visualized by removing the crystal violet.
The five cell lines infected with VTT and the mutants, and the viruses were harvested after 96 h and titrated in BHK-21 cells. Titers of the mutants in different cell lines were determined by plaque assay and expressed as plaque-forming units (pfu)/ml virus suspension
For the rabbit skin pathogenicity assay, three concentrations (106, 107, and 108 pfu in 0.1 ml sterile PBS) of MVTT1, MVTT2, MVTT3, or VTT were injected intradermally on the shaved backs of rabbits. Each concentration was performed in three rabbits. The diameters of erythema resulting from inoculation were measured daily for 18 d.
Mutant virulence was evaluated by measuring the weight loss of inbred BALB/c mice after viral inoculation, according to a previously described method
Twelve groups of 3-week-old female BALB/c mice (n = 6) were inoculated intracranially with 105, 106, or 107 pfu of MVTT1, MVTT2, MVTT3, or VTT in 10 µl of sterile PBS and deaths were recorded daily for 14 d. ICLD50's were calculated using the Reed–Muench method
Groups of 6-week-old female BALB/c mice (n = 10) were injected intramuscularly (i.m.) with 5×104 PFU/mouse of MVTT1, MVTT2, MVTT3 or VTT in 0.1 ml PBS, or mock infected with PBS. Three weeks later, sera were taken from caudal vein and each mouse received a booster of the same dose. Two weeks later, the animals were euthanized and samples of heat-inactivated sera were processed for mouse IL-2, IL-4, IL-10 or IFN-γ ELISA analysis using commercially kits (Groundwork Biotechnology Diagnosticate, USA).
To determine the
Heat-inactivated mouse serum were serially diluted in twofold steps, mixed with the parental VTT virus strain at a concentration of 100 PFU per well, and transferred to a monolayer of BHK-21 cells. 96 h post-incubation, BHK-21 cells were inspected for cytopathic effects. IC50 were determined by the highest dilution of mouse serum that generated 50% viral plaque reduction and was calculated by the method of Reed and Muench
TC7L-TK2L and TA35R genes were deleted in VTT genome to generate three mutants (
(A) The TC7L-TK2L and TA35R genomic deletions were found in the viral genome. (B) EGFP was expressed by mutants MVTT1-EGFP, MVTT2-EGFP, or MVTT3-EGFP in the BHK-21 cells. The virus-infected cells were visualized by isolated fluorescent plaque, which was recognized in the same visual fields. Non-fluorescent plaques appeared because of recombinant MVTT1, MVTT2, or MVTT3 in the BHK-21 cells. All photos were taken at 200× magnification.
Diagnostic PCR was performed to identify the final mutant. The deletion of the TC7L-TK2L and TA35R genes was investigated by PCR 72 h after infection of BHK-21 cells with 0.01 MOI of wild-type VTT. Approximately 366 bp (TC7L-TK2L; A, lane 1) and 353 bp (TA35R; A, lane 3) were detected by ethidium bromide staining. In addition, the TC7L-TK2L and TA35R fragments were missing in the mutants MVTT1-EGFP (TC7L-TK2L; A, lane 2), MVTT2-EGFP (TA35R; A, lane 4), and MVTT3-EGFP (TC7L-TK2L and TA35R; A, lane 5 and lane 6). The genes 431 bp flanking the TC7L-TK2L regions (B, MVTT1, lane 1; and MVTT3, lane 3) and the genes 1142 bp flanking the TA35R regions (B, MVTT2, lane 2; and MVTT3, lane 4) were investigated to identify non-EGFP-expressing virus. Evaluation of the genetic stability in the BHK-21 cells after 2, 4, 6, 8 and 10 passages. TC7L-TK2L gene of MVTT1 (C, lanes 2–6), TA35R gene of MVTT2 (D, lane 2–6), TC7L-TK2L gene of MVTT3 (G, lanes 3–7), and TA35R gene of MVTT3 (G, lanes 8–12) produced negative results, comparing to positive PCR results. The genes of MVTT1 flanking the TC7L-TK2L regions (E), the genes of MVTT2 flanking the TA35R regions (F), and the genes of MVTT3 flanking the TC7L-TK2L regions (H, lanes 1–5) or TA35R regions (H, lanes 6–10) were detected correctly.
Clones of purified non-fluorescent plaque in which TC7L-TK2L, TA35R, and both TC7L-TK2L and TA35R were deleted were identified (
The three mutants were added to cultures of BHK-21 cells passaged 10 times at 1 day intervals to determine their stability. The three genomic DNAs were then analyzed for the presence of TC7L-TK2L and TA35R. The mutant MVTT1 could not transcribe the gene within TC7L-TK2L after 2, 4, 6, 8, and 10 passages in BHK-21 cells, contrary to the result of VTT (
The MTT colorimetric assay was performed to detect viabilities of the five cell lines after infection (
Effects of MVTT1, MVTT2, MVTT3, or VTT (0.05 PFU/cell) and infection times on the cell viabilities of HeLa (A), MDCK (B), PK(15) (C), BHK-21 (D), and Vero (E). Cells were seeded in 96-well plates (1×104 cells/well) one day before they were infected with 0.05 PFU/cell of MVTT1, MVTT2, MVTT3, or VTT. Cell viability was measured daily for 4 d by MTT colorimetric assay. All measurements were performed in triplicate. Data are presented as mean ± SD.
Confluent monolayers of PK(15), MDCK, HeLa, BHK-21 and Vero cells in 12-well plates were infected with 0.05 PFU/cell of MVTT1, MVTT2, MVTT3, or VTT viruses. The plates were incubated at 37°C for 2 d prior to staining with 0.1% crystal violet. The pathogenicity of MVTT1, MVTT2, and MVTT3 apparently decreased in all five cell lines (compared with VTT as the control).
The five mammalian cell lines were infected with 0.05 PFU/cell of each virus. Based on crystal violet staining, each cell type tested was found to be infected by VTT. Compared with VTT, MVTT3 apparently did not produce cytopathic effect in PK(15), MDCK, HeLa, and Vero cells (
All three mutants proved capable of growing in the BHK-21 cells. The titration was shown in
PK(15), MDCK, HeLa, BHK-21 and Vero cells infected with 0.05 MOI of VTT and the mutants, and then the viruses were harvested and titrated in BHK-21 cells. Virus titer was determined by measuring the plaque assays.
Cutaneous lesion is a side effect of the virus inoculation; therefore, virulence was further assessed by intradermal inoculation on the rabbit dorsal spine. The lesion diameters were measured 18 d after inoculation. The diameters of the central lesions normally reached their peak on day 7. The diameters when the erythemas reached their peak are presented in
Each rabbit was inoculated intradermally on the dorsal spine with a 10-fold dilution of MVTT1, MVTT2, MVTT3, or VTT (106, 107, and 108 pfu). The central size diameters were recorded 18 d after inoculation. The diameters when erythemas reached their peak for each rabbit (A and C) are presented. The average difference is shown between lesion diameter produced by each recombinant and that by VTT (B). The data represents the mean ± SD of three lesions. The mean lesion sizes produced by MVTT1 and MVTT2 were significantly smaller than those formed by parental VTT. Lesions did not develop after inoculation with MVTT3 at the three doses.
Groups of six mice were infected with 105, 106, or 107 PFU of MVTT1, MVTT2, and MVTT3 deletion mutants or VTT intranasally, and weight loss was recorded daily. As shown in
Groups of mice (n = 6) were infected intranasally with different doses of MVTT1, MVTT2, MVTT3,VTT, or PBS on day 0, at doses of 105 pfu (A), 106 pfu (B), and 107 pfu (C). The mean group weight was expressed as the percentage of the mean body weight change. The error bar indicates the standard deviation of each group. Each dose group of VTT and mutant showed a significant difference in weight change (P<0.05). No mice in the MVTT3 and PBS group lost weight or showed signs of illness.
Six 3-week-old BALB/c mice were infected via the intracranial route to evaluate the neurovirulence of the three recombinant strains (
Six 3-week-old mice in each dilution group were inoculated intracranially with 105, 106, and 107 pfu of MVTT3 and VTT (A), and MVTT1 and MVTT2 (B). The survival rate of animals were observed for 14 d. All mice inoculated with MVTT1, MVTT3 and105 pfu of MVTT2 survived. All mice infected with 106 and 107 pfu of MVTT2 and VTT died.
Delivery of MVTT1, MVTT2, MVTT3 and VTT elicited strong systemic immune responses induced by i.m. vaccination. IL-2, IL-4, IL-10 and IFN-γ responses, as measured by mouse ELISA, were already detectable using a spectrophotometer (
Ten 6-week-old BALB/c mice received 5×104 PFU MVTT1, MVTT2 or MVTT3 by intramuscular route and three weeks later received booster of the same dose in 0.1 ml of PBS. The serum were harvested on weeks 3 and 5 post infection (i.m.). Shown were induced IL-2, IL-4, IL-10 and IFN-γsystemic immune responses in the two vaccinated cohorts as measured by mouse ELISA kit analysis. All measurements were performed in triplicate. Data were presented as mean ± SD.
Neutralizing antibody titer against the parental vector VTT was shown in
Murine sera collected at 3 and 5 weeks after infection. Neutralization antibody titer was calculated by determining the highest serum dilution to generated 50% viral plaque reduction. All measurements were performed in triplicate. Data were presented as mean ± SD.
The protective immunogenicities of 5×104 PFU MVTT1, MVTT2 and MVTT3 were determined by using a mouse model challenged with a highly pathogenic VTT strain. The mice immunized with MVTT1, MVTT2 and MVTT3 did not exhibit any significant differences in weight post challenge and no signs of illness. In contrast, beginning at the week of infection, all the mice in the PBS group clearly showed clinical signs of disseminated disease, such as ruffling fur and arched back. All mice immunized of MVTT1, MVTT2 or MVTT3 survived, whereas all sham-immunized mice were killed from 7 days because of a 30% weight loss (
Groups of ten BALB/c mice (3-week-old) were vaccinated with 5×104 PFU MVTT1, MVTT2 or MVTT3 via intramuscular routes. Mice received PBS were included as controls. Four weeks post-immunization, the mice were challenged intranasally with 500×LD50 VTT strain. Cross marks indicate the mice that were killed because of a 30% weight loss. Data show average percent change in pre-challenge weigh. The error bar indicates the standard deviation (SD) of animals from each group.
VTT was used as a vaccine against smallpox in China for millions of people, but remains neurovirulent in mice
The phenotypic changes observed in mutants were related to the loss of the TC7L-TK2L and TA35R genes in the VTT genome. As previous study shows, the loss of M1L-K2L genes reduced cell-to-cell spread including HeLa, PK(15), MDCK and Vero cells
Vaccine candidates not only had strongly diminished virulence, but also maintaining good immunogenicity. Both MVA and NYVAC gave roughly comparable levels of protection against pathogenic vaccinia
We thank Ping Li for the valuable advice on cell culture. We also thank Bo Hu and Shouwen Du for their technical support during the animal tests.