Generation of the mutants
TC7L-TK2L and TA35R genes were deleted in VTT genome to generate three mutants (). Plaques containing mutants were recognized by their fluorescence, and mutants were clonally purified in the BHK-21 cells by repeated plaque isolation (). showed that the double deletions 366 bp (9,357–9,732) of TC7L-TK2L and 353 bp (139,162–139,515) of TA35R were successful based on the PCR results for the plaque-purified EGFP virus.
Schematic representation of the MVTT3 genome and identification of mutants.
PCR analysis results of the TC7L-TK2L and TA35R genes of the isolated mutant.
Clones of purified non-fluorescent plaque in which TC7L-TK2L, TA35R, and both TC7L-TK2L and TA35R were deleted were identified (). 431 bp (14,886–25,471) correct viral sequences flanking the deletion sites of TC7L-TK2L and 1142 bp (137,883–139,679) correct viral sequences flanking the deletion sites of TA35R were confirmed by nucleotide sequencing after removal of EGFP. showed that the double deletions were successful based on the PCR results for the generation of three deletion viral mutants from VTT, namely, MVTT1, MVTT2 and MVTT3.
Genetic stability of the mutants
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 (). Similar results were obtained with MVTT2 and MVTT3 (). The correct 431 bp and 1142 bp viral sequences flanking the deletion sites of TC7L-TK2L and TA35R respectively were confirmed by nucleotide sequencing after 2, 4, 6, 8, and 10 passages. These findings indicate that the mutants were genetically stable after many passages (). This characteristic was crucial for eliminating the non-essential genes in developing a lower virulence vector for human use.
Inhibition of cell growth by the mutants
The MTT colorimetric assay was performed to detect viabilities of the five cell lines after infection (). When cells were treated with the four viruses, MVTT3 had little effect on cell viability. In contrast, MVTT1, MVTT2 and VTT inhibited growth by 5%–50%, 45%–55% and 60%–80% after 96 h, respectively; growth of cells treated with VTT was eventually completely blocked. Therefore, the survival rates of the five cell lines infected with VTT were significantly lower than those infected with the mutants. The survival rate of cells infected with MVTT3 was higher than those infected with the other two mutants. The survival rate of cells infected with MVTT1 was higher than that of cells infected with MVTT2. Similar results were obtained by tests using crystal violet staining of the five cell lines infected with the mutants ().
Assessment of growth of five cell lines infected with the mutants.
Plaque phenotypes formed by infection with mutant viruses.
Mutants spread efficiently in five cell lines
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 (). The MVTT3 virus, however, displayed lower spread than MVTT1 (). The spread of MVTT1 evidently decreased than MVTT2, and MVTT2 displayed lower spread than VTT in all five cell lines, indicating that deletion of TC7L-TK2L further attenuated the virus (). These results also indicate that the loss of viral genomic fragments TC7L-TK2L or TA35R may not spread efficiently in tissue culture.
Grown of mutants
All three mutants proved capable of growing in the BHK-21 cells. The titration was shown in . The titers of the mutant viruses in each cell were lower than that of the parental virus. The titer of MVTT3 was lower than MVTT2 and MVTT1.
Virus titer of mutants in five cell lines tested.
Deletion of the TC7L-TK2L or TA35R gene attenuates VTT virulence in rabbits
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 . The average differences in largest lesion diameter caused by each mutant were compared against that produced by VTT (). The largest lesion size for each rabbit was shown in . Rabbit skin remained intact when inoculated with varied doses of MVTT3, whereas the rabbit immunized with VTT developed a severe rash even when administered a dose of 106 pfu. This was similar to the result of inoculation of 108 pfu MVTT1. No significant difference between the lesions formed by MVTT1 and MVTT2 were observed (P>0.05). Recovery from skin lesions caused by VTT was slower than recovery caused by MVTT1 and MVTT2. The extent of recovery from lesions caused by MVTT1 was better than recovery from that caused by MVTT2.
Characterization of virulence of strains intradermally injected in rabbits.
MVTT3 is safe for BALB/c mice
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 , all mice survived during a period of 25 d. We show the first 11days after infection, which has significant differences between mutants group and VTT group. In contrast to the mice that received PBS, mice infected with MVTT1, MVTT2, or MVTT3 deletion mutants showed mild signs of illness, and mice in the MVTT3 group gained weight more quickly than did the mice in the other two groups. However, in contrast to VTT, there was an evident positive correlation between the dose of virus inoculation and the body weight loss. The VTT group had more severe signs of illness, and weight loss reached 12.19% on day 9. The lowest dose of 105 pfu of VTT resulted in a significant body weight loss. The three mutants were likely attenuated by at least about 100-fold. The weight gain rate of MVTT3 was similar as PBS group. The weight gain rate of MVTT3 was higher than MVTT1 and MVTT2. These results suggest that MVTT1, MVTT2, and MVTT3 viruses were attenuated with respect to its parent VTT, and that the virulence of MVTT3 was less than that of the other strains. This experiment was repeated twice with similar results obtained.
Virulence of MVTT1, MVTT2, and MVTT3 in mice after intranasal inoculations.
In vivo virulence of MVTT3
Six 3-week-old BALB/c mice were infected via the intracranial route to evaluate the neurovirulence of the three recombinant strains (). Since the ICLD50 of MVTT2 was 2.5×105 pfu and that of VTT was 3.1×103 PFU, MVTT2 was significantly attenuated by 80-fold. No mortality was observed in the mice infected with MVTT1 and MVTT3 although they were given the highest dose of 107 pfu. These indicate that MVTT1 and MVTT3 were attenuated by at least 3,200-fold relative to the ICLD50 of VTT. The two mutants were essentially non-neurovirulent. These data indicate that the MVTT2 mutant exhibited attenuated but to a lesser degree than the MVTT1 and MVTT3 mutants. The results of the ICLD50 were consistent with those from the intranasal infection experiment. This experiment was repeated twice with similar results obtained.
Protection from lethal challenge.
Immunogenicity of mutants
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 (). Mice were infected and on week three and five serum were harvested for IL-2, IL-4, IL-10 and IFN-γ production. The groups of MVTT1, MVTT2, MVTT3 and VTT, after twice administration, remained robust immune responses than PBS group. IL-10 responses was more difficult to induce. No significant response was seen after second immunization. For monitoring the production of IL-2, IL-4, and IFN-γ, responses were enhanced consecutively in twice i.m.-immunized. The difference among the mutants was not significant.
Immune responses induced by vaccination.
Neutralizing antibody titer against the parental vector VTT was shown in . Immunization of each group induced systemic neutralizing antibody using the dose of 5×104 PFU/mouse. The second immunization marked effects on level of neutralizing antibody. Neutralizing antibody titers of mutants are similar to those with the parental strain (P>0.05). Collectively, these results demonstrated that both mutants and VTT can induce robust systemic immune responses.
Neutralization antibody titer in murine sera.
Mutants protects mice from pathogenic VTT strain challenge
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 (). All three mutants were equally effective in protection of mice against challenge with the VTT strain. These data support the conclusion that a VTT-based vaccine without TC7L-TK2L and TA35R genes would be a efficacious and immunogenic vaccine.
Protection of mice against pathogenic vaccinia VTT strain challenge.