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Construction of an attenuated Tian Tan vaccinia virus strain by deletion of TA35R and TJ2R genes
Accepted Manuscript Title: Construction of an Attenuated Tian Tan Vaccinia Virus Strain by Deletion of TA35R and TJ2R Genes Authors: Yiquan Li, Shuang Chen, Jinbo Fang, Yilong Zhu, Bing Bai, Wenjie Li, Xunzhe Yin, Jing Wang, Xing Liu, Jicheng Han, Xiao Li, Lili Sun, Ningyi Jin PII: DOI: Reference:
S0168-1702(18)30306-X https://doi.org/10.1016/j.virusres.2018.06.017 VIRUS 97437
To appear in:
Virus Research
Received date: Revised date: Accepted date:
18-5-2018 29-6-2018 30-6-2018
Please cite this article as: Li Y, Chen S, Fang J, Zhu Y, Bai B, Li W, Yin X, Wang J, Liu X, Han J, Li X, Sun L, Jin N, Construction of an Attenuated Tian Tan Vaccinia Virus Strain by Deletion of TA35R and TJ2R Genes, Virus Research (2018), https://doi.org/10.1016/j.virusres.2018.06.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Construction of an Attenuated Tian Tan Vaccinia Virus Strain by Deletion of TA35R and TJ2R Genes
Yiquan Li a,b, Shuang Chen a,b,c, Jinbo Fang b,d, Yilong Zhu b,d, Bing Bai b,d, Wenjie Li b,d,e,
a,b,d,f,h*
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Xunzhe Yin b,d, Jing Wang b, Xing Liu b, Jicheng Han a,b, Xiao Li b,d,f*, Lili Sun b,g*, Ningyi Jin
Medical College, Yanbian University, Yanji 133002, P. R. China
b
Institute of Military Veterinary Medicine, Academy of Military Medical Science, Changchun
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a
130122, P. R. China
School of Medical Inspection, Jilin Medical University, Jilin 132013, P. R. China
d
Changchun University of Chinese Medicine, Changchun 130021, P. R. China
e
College of Animal Science and Technology, Guangxi University, Nanning 530005, P. R.
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c
f
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China
Institute of Virology, Wenzhou University Town, Wenzhou 325035, P. R. China Department of Head and Neck Surgery, Tumor Hospital of Jilin Province, Changchun
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g
h
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130012, P. R. China
Jiang su Co-innovation Center for Prevention and Control of Important Animal Infectious
Diseases and Zoonoses, Yangzhou 225009, P. R. China
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*Corresponding author
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Corresponding author: Ningyi Jin, Xiao Li and Lili Sun E-mail: [email protected] (JN); [email protected] (LX); [email protected] (SL)
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Tel: +86-0431-86985923 Fax: +86-0431-87985861 Address: Liuying west road, 666, Jingyue economic & technological development zone, Changchun, Jilin, 130122, P.R.China
Highlights
We construct an attenuated multi-genes deficient vaccinia virus Tian Tan strain.
Replication ability of the attenuated virus is not reduced.
Cell toxicity of the attenuated virus is significantly decreased.
The in vivo virulence of the attenuated virus is reduced.
The attenuated virus is a candidate vector with increased safety.
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Abstract
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rVTT-TA35-TJ, an attenuated vaccinia virus Tian Tan strain (VTT), was constructed by
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knocking out two non-essential gene fragments (TA35R and TJ2R) related to virulence, immunomodulation, and host range; and by combining double marker screening with
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exogenous and endogenous selectable marker knock-out techniques. Here, the shuttle plasmids pSK-TA35 and pSK-TJ were constructed, containing two pairs of recombinant
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arms: early and late strong promoter pE/L and EGFP as an exogenous selectable marker. The recombinant vaccinia virus rVTT-TA35-TJ without exogenous selection markers was then
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obtained through homologous recombination technology and the Cre/loxP system. Knocking out the two gene fragments does not affect the replication ability of the virus and displays a good genetic stability. Furthermore, a series of in vivo and in vitro experiments demonstrate
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that although virulence of rVTT-TA35-TJ is attenuated significantly, high immunogenicity was maintained. These results support the potential development of rVTT-TA35-TJ as a safe viral vector or vaccine. Keywords: Attenuated vaccinia virus vector; Tian Tan strain; virulence; immunogenicity
1. Introduction
The vacinia virus (VV) is a member of the genus Orthopoxvirus, of the family Poxviridae. The vaccinia virus has a genome size of 185 to 200 kb, divided into central conservative areas and flanking areas, and encodes 200 proteins. The highly conserved central part of the genome, which comprises the majority of the VV genome, contains the essential genes that play a critical role in virus replication, such as DNA replication, transcription, and viral particle assembly (Qin et al., 2015). The genes present at both ends of
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the genome are commonly used to identify species or host specificities, and to encode
proteins that regulate the host immune system and virulence factors (Liu and McFadden,
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2015).
Vaccinia virus Tian Tan strain (VTT), a unique vaccinia virus strain in China, has played a key role in the eradication of smallpox in China, but the virus also causes side effects, such
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as fever, fatigue, ocular vaccinia infection, gangrenosa vaccinia, and encephalitis (Lederman
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et al., 2009; Wollenberg and Engler, 2004). These can be severe or even life-threatening,
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especially in patients with immunosuppression, eczema, chronic skin diseases, and heart disease (Bray and Wright, 2003; Casey et al., 2005; Eckart et al., 2004; Kemper et al., 2002).
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Therefore, the safety of vaccinia virus needs further developed.
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As a double-stranded DNA virus, vaccinia virus has unique biological characteristics. First, unlike other DNA viruses, vaccinia virus is present in the cytoplasm during the entire infection cycle from virus entry to progeny virus formation. Therefore, the virus's DNA does
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not integrate into the host's genome. Second, vaccinia virus has a large genome and can accommodate at least 25 kb of foreign genes. In addition, vaccinia virus particles are
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relatively stable and can be stored as dry powder form, making them easy to transport for clinical application. It is precisely due to the above advantages of vaccinia virus that many scientists have tried to use vaccinia virus as a carrier to express antigenic proteins of various
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diseases, and have conducted various vaccine research. Many vaccines have been developed with vaccinia virus as a carrier to prevent other
diseases (Adelfinger et al., 2014; Garcia-Arriaza et al., 2013; Noisumdaeng et al., 2013; Remy-Ziller et al., 2014), such as hepatitis A virus, hepatitis B virus, herpes simplex virus, and human immunodeficiency virus. For example, the genetically engineered vaccine against the rabies virus glycoprotein is synthesized using the VV Copenhagen strain as the vector
(Pastoret and Brochier, 1996), the human cytomegalovirus pp65 protein uses the ALVAC vector, the vesicular stomatitis virus glycoprotein protein uses the VV Western Reserve (WR) strain as the vector (Mackett et al., 1985), and the Newcastle Disease Virus fusion protein uses the VV Elstree strain as the vector (Meulemans et al., 1988). Although significant progress has been made in the study of VV as a vector, there are still some limitations, such as the selection of vaccinia viruses after viral recombination, the insertion and deletion of
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exogenous selection markers, and the complexity of vector construction procedures (MacNeil et al., 2009). Currently, reducing the side effects of VV vaccines, simplifying the preparation
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process, and improving the efficiency of vector vaccines are research hotspots of VV vectors.
Here, we obtained the attenuated strain rVTT-TA35-TJ by knocking out the following two non-essential gene segments of the VTT strain: TA35R (138,881139,570) and TJ2R
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(80,294-80,827). The knockout fragments TA35R contain virulence-related genes and
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immunomodulation-related genes. NYVAC, as one of the most successful gene-knockout
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attenuated VV vectors, was obtained by phenotypic attenuation after knockout of the 18 ORFs, containing the TA35R gene that is a highly expressed gene responsible for the
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formation of A-type inclusion bodies (Tartaglia et al., 1992). The production of the thymidine
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kinase (TK) gene of vaccinia virus can be inhibited by using deoxyuridine-like (Brdu) to block the synthesis of the viral nucleic acid, inhibiting the replication of vaccinia virus in the cell, allowing for selection of the vaccinia virus lacking the TK gene. The TK of VTT is
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encoded by the TJ2R gene segment in the genome which can be used as an endogenous selection marker for the screening of recombinant vaccinia virus.
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To further simplify the procedure of recombinant vaccinia virus construction, the
screening process was optimized: a double labeling screening method using both exogenous selection marker enhanced green fluorescent protein (EGFP) and an endogenous selection
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marker TK (TJ2R), and knockout of the exogenous selection marker was achieved by introducing the Cre/LoxP system into the shuttle vector plasmid (Rintoul et al., 2011). These strategies significantly shorten the cycle of recombinant vaccinia virus and improve the efficiency of screening for recombinants. Subsequent in vitro and in vivo experiments in mice and rabbits further examined the recombinant vaccinia virus lacking TA35R and TJ2R from vaccinia Tian Tan strain in relation to its virulence and immunogenicity.
2. Materials and Methods 2.1 Cells, viruses, and animals BHK-21, MDCK, Hela, Vero, and PK-15 were purchased from the China Center for Type Culture Collection (CCTCC). These cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Hyclone, Beijing, China) with 10% fetal bovine serum (FBS) (Hyclone, Beijing, China) and penicillin (10,000 U/mL)/1% streptomycin (10 mg/mL) (Hyclone,
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Beijing, China). The VTT strain (GenBank accession no. AF095689) was obtained from the Institute of Virology at the Chinese Center for Disease Control and Prevention.
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Three-week-old and six-week-old BALB/c male mice and New Zealand male white rabbits were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences of China.
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The animal experimental protocols were approved by the Institutional Animal Care and
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Use Committee (IACUC) of the Chinese Academy of Military Medical Science, Changchun,
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China (10ZDGG007). All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
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2.2 Construction of VTT shuttle plasmid
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To construct the genes deleted VTT, TA35 (138,881139,570) and TJ (80,294-80,827) were selected as deletions based on results of whole-genome sequence analysis of the VTT strain (unpublished data). The shuttle plasmid pSK-TA35 containing TA35 gene recombinant
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left arm (138,353 – 138,881 bp)-LoxP-PE/L-EGFP-T5NT-LoxP, TA35 gene recombinant right arm (139,570 – 140,607 bp), and shuttle plasmid pSK-TJ containing TJ gene left
arm
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recombinant
(79,784–80,294
bp)-Loxp-PE/L-EGFP-T5NT-Loxp,
TJ
gene
recombinant right arm (80,827–81,855 bp) sites were synthesized from Shanghai Generay Biotech Co., Ltd of China.
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2.3 Construction and genetic stability analysis of gene deletion VTT The recombinant virus rVTT-A35-J-EGFP, in which the TA35 and TJ genes were
replaced with EGFP, was constructed by homologous recombination. rVTT-TA35-TJ, lacking the TA35 and TJ genes, was obtained from rVTT-A35-J-EGFP, following the knockout of EGFP by Cre/Loxp system. BHK-21 cells were infected with VTT at a MOI of 0.1 for 2 h. Then, the cells were transfected with the mixture of the shuttle pSK-TJ and Effectene
Transfection Reagent (QIAGEN). Following culture for 72 h, the recombinant virus was released by three freeze-thaw cycles and used for further infection. Under an inverted fluorescence microscope, green fluorescent plaques were picked out, and this purification process was repeated six times until the monoclonal fluorescent plaque (i.e., the recombinant virus rVTT-J-EGFP) was purified. rVTT-J-EGFP and the plasmid pVAX-Cre were co-transfected into BHK-21 cells, and rVTT-J lacking the TJ gene was isolated by screening
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the non-green fluorescent plaques (i.e., the non-EGFP-expressing virus) under a fluorescence microscope; this step was also repeated six times. The TA35 gene was knocked out in the
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same manner. Ultimately, both the TA35 and TJ genes were deleted, and the vector was named rVTT-TA35-TJ.
rVTT-TA35-TJ was identified by PCR. The genome of rVTT-TA35-TJ was extracted to
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use as template for amplifying the partial segments TA35 (307 bp) and TJ (261 bp) by PCR.
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The primers are as follows: TA35-1F; CAGCGTGATTCTTACCAGATATT, TA35-1R;
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TGTTGCGAGCATTACTGCGTTTA, TJ-1F; GATTGGAAGCAACTAAACTATGT, TJ-1R; CCTCCTATTATTTCTATCTCGGT. At the same time, we also designed primers on both ends
follows:
TA35-2F;
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The primers are as
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of the recombination arm and amplified fragments TA35 (751 bp) and TJ (551 bp) by PCR.
ACTGGACGGCGTCCATT,
TJ-2F;
AATGTCCAGGATTCTCCTA, TA35-2R; AGTGAACAATAATTAATTC,
TJ-2R;
AATTTAATCAATGTTTAT. The PCR reaction conditions were 95°C 5 min, 30 cycles of
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95°C 30 s, 57°C 30 s, 72°C 30 s, and a final extension at 72°C for 10 min. The genome of VTT was used as a control.
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The genomes of the 5th, 10th, 15th, and 20th passage in rVTT-TA35-TJ-infected
BHK-21 cells were extracted to detect genetic stability of rVTT-TA35-TJ by PCR. The genome of VTT was used as a control.
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2.4 Characterization of rVTT-TA35-TJ The characterization of rVTT-TA35-TJ was performed by growth curve, plaque
formation assay, and MTS assay. To detect and observe the formation of virus plaques of rVTT-TA35-TJ-infected cells, the BHK-21, MDCK, Hela, Vero, and PK-15 cells were infected with rVTT-TA35-TJ and VTT at 0.5 MOI for 48 h. Then, the cytopathic effects of these infected cells were observed with a fluorescence microscope.
To study the multiplication of rVTT-TA35-TJ, BHK-21, MDCK, Hela, Vero, and PK-15 cells were infected with rVTT-TA35-TJ and VTT at 0.5 MOI. Then, the viral titer at 3, 12, 24, and 48 h was determined. BHK-21 cell was cultured in 24-well cell culture plates for 24 h. A serial 8-fold serial dilution of the rVTT-TA35-TJ and VTT was performed. The cell culture medium was discarded, and then the virus was inoculated at a virus dose of 200 ul/well, with 4 repeat wells for each dilution. Incubate at 37 °C, 5% CO2 for 5 days. Calculation formula:
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PFU = (average number of virus plaques × dilution) / inoculum volume. The growth curves were then extrapolated from the viral titers at each time point.
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To assess the virulence of rVTT-TA35-TJ, BHK-21, MDCK, Hela, Vero, and PK-15 cells were infected with rVTT-TA35-TJ and VTT at 0.5 MOI and then cultured at 37°C with 5% CO2. The virus-infected cells were cultured for 2 h following the addition of 20 μl of MTS
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solution (Promega) at 24, 48, 72, and 96 h post infection. The number of viable cells was
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quantified by measuring optical density (OD) at 490 nm. Cell viability was calculated
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according to the following formula: 100 × (absorbance of culture in the treated
2.5 Safety analysis of rVTT-TA35-TJ
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wells/absorbance of culture in the control wells) (Li et al., 2006; Mosmann, 1983).
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The safety of rVTT-TA35-TJ was evaluated by detecting neurotoxicity, measuring weight changes in mice, and measuring pock size in New Zealand white rabbits. Six-week-old male BALB/c mice were divided into 7 groups (n = 10). Group 1 received 1×105 PFU of
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rVTT-TA35-TJ in 20 μl of normal saline by intranasal inoculation, group 2 received 1×10 6 PFU of rVTT-TA35-TJ, group 3 received 1×107 PFU of rVTT-TA35-TJ, group 4 received
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1×105 PFU of VTT, group 5 received 1×106 PFU of VTT, group 6 received 1×107 PFU of VTT, and group 7 received 20 μl of normal saline. The body weight of each mouse was recorded every day for 25 days post inoculation.
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Three-week-old male BALB/c mice were divided into 7 groups (n = 10) and inoculated intracranially with 10 μl of rVTT-TA35-TJ and VTT diluted with normal saline at doses of 1×105 PFU/10 μl PBS, 1×106 PFU/10 μl PBS, or 1×107 PFU/10 μl. Grouping arrangement was identical as above; the control group was inoculated with PBS. Deaths were observed for 14 d post inoculation. The intracranial 50% lethal infectious dose (ICLD50) was calculated using the Reed–Muench method (Reed LJ, 1938).
The backs of male New Zealand white rabbits received 1×105 PFU/100 μl of rVTT-TA35-TJ and VTT, 1×106 PFU/100 μl of rVTT-TA35-TJ and VTT, or 1×107 PFU/100 μl of rVTT-TA35-TJ and VTT by intradermal injection. Normal saline was used as a control group. Every concentration was injected into two rabbits and repeated three times. The pock size was measured by Electronic vernier caliper daily for 18 d post injection. 2.6 Immunogenicity of rVTT-TA35-TJ in mice
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The immunogenicity was estimated by detecting the levels of VTT-specific antibody, IL-2, IL-4, IL-10, IFN-γ, and neutralizing antibody titer. Six-week-old BALB/c mice (n = 10)
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were inoculated intramuscularly with 1×106 PFU/ml normal saline with rVTT-TA35-TJ and VTT; the control group of goats was inoculated with normal saline. The sera separated from the weekly blood samples were used for the detection of IL-2, IL-4, IL-10, and IFN-γ with
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ELISA kits (MYBioSource) after vaccinating with rVTT-TA35-TJ. The levels of
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VTT-specific IgG were measured by ELISA. The purified VTT was diluted to 1×106 PFU/ml. The virus was diluted 10 times with the coating solution, and then added to the ELISA plate
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at 100 μL/well, and stay overnight at 4℃. Discard the coating solution, wash the plate 3
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times, and Add 200 μL blocking solution per well and incubate at 37°C for 2 h. Wash the plate 5 times and Add 100 μL of 10-fold diluted serum sample to each well and incubate for 2
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h at 37°C. Wash the plate 5 times and Add 100 μL of HRP-labeled antibody to each well and incubate for 2 h at 37°C. Wash the plate 5 times and Add 100 μL 1×TMB solution to each
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well and incubate at room temperature for 15 min. Add 50 μL of stop solution per well. And the optical density (OD) was detected at 450 nm. The neutralization assay was performed as
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described previously (Kan et al., 2012). The results were calculated using the method of Reed and Muench (Reed LJ, 1938). 2.7 Statistical analysis
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The statistical analysis was performed using data from at least three independent
experiments using the Statistical Package for the Social Sciences (SPSS) statistical software package (version 15.0; SPSS, Inc., Chicago, IL, USA), and the results were obtained using GraphPad Prism version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA). Student’s t-tests were performed for all analyses and differences among groups were determined by one-way ANOVA with Tukey's post hoc test.
P < 0.05 was considered to indicate statistical
significance. Data are presented as the mean ± standard deviation (SD).
3. Results 3.1 Construction and identification of rVTT-TA35-TJ The shuttle plasmids pSK-TA35 and pSK-TJ were identified by double-restriction enzyme digestion with EcoRI and PstI. The EGFP fragment (720 bp) was obtained by double
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digestion of the shuttle plasmids. These results indicate that the two shuttle plasmids were constructed successfully.
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The screening process and PCR results for rVTT-TA35-TJ are shown in Figure 2A-E. From Figure 2C we can see that the 261 bp and 307 bp fragments cannot be amplified from
the rVTT-TA35-TJ genome, but can be amplified from the VTT genome; and from Figure
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2D-E we can see that the fragments TA35 (751 bp) and TJ (551 bp) can be amplified from the
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VTT genome, but cannot be amplified from the rVTT-TA35-TJ genome demonstrating that
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the partial segment of TJ and TA35 genes was knocked out.
rVTT-TA35-TJ was continuously passaged 20 times, and the PCR products of TA35 (307
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bp) or TJ (261 bp) of the genome from the 5th, 10th, 15th, and 20th passage in VTT-infected
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BHK-21 cells could be amplified. However, PCR amplification of the 5th, 10th, 15th, and 20th generation of the rVTT-TA35-TJ genome under the same conditions did not produce the corresponding bands. These results demonstrate that rVTT-TA35-TJ has good genetic
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stability (Fig. 2F-G).
3.2 Characterization of rVTT-TA35-TJ
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The growth curve results of rVTT-TA35-TJ and VTT are shown in Figure 3. The two
viruses have similar growth curves in the same cells and no significant difference in titers of rVTT-TA35-TJ with VTT was observed, indicating that the deletion of TA35 and TJ gene
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reduce viral titer without affecting normal replication of virus. The cytotoxicity of rVTT-TA35-TJ and VTT was measured by MTS, and the results are
shown in Figure 4A-D. The number of living cells decreased with time, and in the BHK, Vero, and PK-15 cells, the survival rate of VTT-infected cells was significantly lower than the rVTT-TA35-TJ-infected cells at 48 and 72 h (P < 0.01). At 96 h, the survival rate of VTT-infected cells was significantly lower than the rVTT-TA35-TJ-infected cells. These
results suggest that the deletion of TA35 and TJ gene in rVTT-TA35-TJ significantly reduced the in vitro virulence of VTT. Crystal Violet staining demonstrates that plaque formation was clearly observed in all five VTT-infected cell lines, but infection with rVTT-TA35-TJ only formed detectable plaques in BHK-21, Hela, and Vero cells. Furthermore, the number of plaques was significantly smaller than in VTT-infected cells (Fig. 4F). The virus weakened the cytopathic effects of the other
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two cells, similar to the results of MTS, indicating that the deletion of the two fragments
significantly reduces the virulence of VTT. These data suggest that rVTT-TA35-TJ is a safer
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vector for vaccination than the wild-type VTT. 3.3 Weight changes in mice and lesion formation in rabbits post infection
Rabbits were inoculated intradermally with rVTT-TA35-TJ and wild type VTT. Each
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concentration was injected into two rabbits and repeated three times. The size of the pock was
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measured by Electronic vernier caliper daily for 18 d. As shown in Figure 5, the pock sizes of
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rVTT-TA35-TJ in every group and low-dose VTT group reached a maximum on the first day post infection, while middle- and high-dose VTT groups reached maximum pock size on the
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fifth and third day post infection. The pock lesions caused by VTT started to fester from the
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third day and gradually formed scabs. By the tenth day, an obvious scab could be observed. At the end of the experiment, the size of the pock lesions induced by VTT were reduced, but they had not all disappeared. In contrast, the pock lesions induced by middle- and high-dose
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rVTT-TA35-TJ groups started to fester from the fourth day, and gradually formed a scab, with the skin lesions disappearing on the ninth day. The size of pock lesions formed by
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rVTT-TA35-TJ infection at the lowest viral titer were significantly smaller than that of lesions formed by VTT infection at the medium and highest titer (P < 0.05). These results demonstrate that the absence of the deleted gene segments led to a reduction in the virulence
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of rVTT-TA35-TJ in small animals. The rVTT-TA35-TJ and wild type VTT viruses were inoculated intranasally into
6-week-old male BALB/c mice, and body weights were recorded every day for 25 days. As shown in Figure 6A, the weight of mice infected with the highest dose of VTT decreased quickly from the fourth day to the ninth day post inoculation (an average reduction of 23%) and started to gradually increase on the tenth day. Until the end of the experiment, the mean
body weight of the VTT group was lower than other groups. The middle-dose VTT group displayed similar results to the high-dose VTT group. However, the low-dose VTT group demonstrates a slowly rising trend. The three rVTT-TA35-TJ groups displayed a similar trend to the PBS control group. In the three rVTT-TA35-TJ groups, the high-dose group incurred more weight loss than the other two groups (P < 0.05). The rVTT-TA35-TJ and wild type VTT viruses were intracranially inoculated into
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3-week-old male BALB/c mice, and the deaths were observed for 14 days post inoculation. From the first day, the VTT groups showed strong neurological symptoms such as lassitude,
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scruffy fur, convulsions, and stiffness. However, the rVTT-TA35-TJ groups did not exhibit such symptoms. Furthermore, mice inoculated with the three different doses of rVTT-TA35-TJ displayed 100% survival (Fig. 6B), which was significantly higher than the
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survival rate of the VTT-infected mice (P < 0.05). The survival rate of the high dose VTT
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group was 0% on the fourth day post inoculation, and the survival rate of low- and
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middle-dose VTT group was 0% at the sixth and eighth day post inoculation. The ICLD50 of VTT-inoculated mice was 1.16 × 104 PFU. These results suggest that the absence of the
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deleted gene segments in rVTT-TA35-TJ significantly reduces the neurotoxicity of the virus
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in mice. The safety of the recombinant vaccinia virus in mice was thus improved by knocking out two genes, which increases its potential as a vaccine vector. 3.4 Cellular and humoral immunity induced by rVTT-TA35-TJ
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The levels of IL-2, IL-4, IL-10, and IFN-γ were measured in all the mice groups with mouse IFN-γ ELISA kit on day 14 and 35 post immunization. These results indicate that the
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levels of IL-2, IL-4, IL-10, and IFN-γ in serum of the rVTT-TA35-TJ and VTT immunized groups were significantly higher than that in the normal saline control group post immunization (Fig. 7A-D, P < 0.01). In contrast, no significant differences were observed
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between the immunization groups (P > 0.05). These results suggest that rVTT-TA35-TJ can induce high levels of IL-2, IL-4, IL-10, and IFN-γ, and maintain its immunogenicity as a vaccine. The mice were inoculated with rVTT-TA35-TJ and VTT at a dose of 106 PFU to detect VTT-specific antibody and neutralizing antibody in the serum. These results are summarized in Figure 7E and 7F. The level of VTT-specific antibody in these immunization groups was
raised on the 14th day post immunization, but it did not significantly increase from day 14 to 28. However, VTT-specific antibody levels also increased at day 35 post immunization. The level of VTT-specific antibody in the immunization groups was significantly higher than in the normal saline control group post immunization (P < 0.01). In contrast, no significant differences were observed between the immunization groups (P > 0.05). The trend of neutralizing antibody titers of the rVTT-TA35-TJ and VTT groups were identical to the trend
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of VTT-specific antibody. Therefore, rVTT-TA35-TJ can stimulate a strong humoral immune
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response.
4. Discussion
The vaccinia virus is relatively safe compared to the human poxvirus, but it inevitably
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causes a series of adverse reactions following the vaccination (Cono et al., 2003; Jacobs et
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al., 2009; Lane and Goldstein, 2003). Although the probability of serious adverse reactions is
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small, its safety still needs to be improved. When vaccinia virus is used as a vaccine vector, severe necrotizing vaccinia can be induced in immunodeficient patients. Therefore, it is
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particularly important for treatment of attenuated vaccinia virus. Currently, the most widely
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used method is to perform gene insertion, deletion, or blockage at a specific position in the viral genome by gene manipulation to obtain an attenuated viral vector. Scientists have been studying the attenuation of Tian Tan strain vaccinia virus for more than 20 years. Wang et al.
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(Wang et al., 2012) established a strain of attenuated Tian Tan strain by knocking out the E3L gene and confirmed good genetic stability. Another study shows that the removal of the viral
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M1L-K2L genes (MVTT2-GFP) was less virulent than VTT by 340-fold, based on the ICLD50 value in mice (Zhu et al., 2007). Furthermore, the removal of the viral C2L to F3L genes (MVTTZCI) attenuated the virus by 1000-fold (Yu et al., 2010).
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In this study, the replication-non-essential gene TJ2R and TA35R of the vaccinia virus
Tian Tan strain was knocked out. The EGFP fluorescent fragment was designed as an exogenous selection marker and TK(TJ2R) was designed as an endogenous selection marker. The Cre/loxP system was introduced into the shuttle plasmid, and the exogenous selection marker was then knocked out. Following continuous screenings and identification, we successfully constructed the TJ2R and TA35R gene deletion VTT attenuated strain:
rVTT-TA35-TJ. The vaccinia virus A35R gene is located in the viral genome at 144 462 to 144 992 bp, is both a virulence and an immunosuppression gene and encodes a 20 kDa protein. Studies have shown that deletion of the A35R gene from the vaccinia virus WR strain genome reduces the virulence approximately 1000 folds compared to the wild virus (Roper, 2006). A35R can prevent antigen presentation and inhibit MHC class II molecules presentation of antigens to
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CD4+ T cells. Following the knock out of the A35R gene, we can increase the immunogenicity of vaccinia virus without affecting the replication ability. Immunization of
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mice with the attenuated virus strain can play a protective role in the wild-virus attack (Rehm and Roper, 2011). Therefore, knocking out the A35R gene reduces the virulence of the
vaccinia virus and increases the immunogenicity, which allows to screen out more attenuated
this
study,
MTS
experiments
demonstrate
that
the
survival
rate
of
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In
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vaccine vectors.
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rVTT-TA35-TJ-infected cells in BHK-21, PK-15, and Vero cells was significantly higher than VTT-infected cells starting at 48 h post infection. Furthermore, and as the infection
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progresses, the differences in survival rate become greater. In Hela and MDCK cells, the
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survival rate of rVTT-TA35-TJ-infected was significantly higher than that of VTT-infected at 96 h post infection. Crystal violet staining reveals identical results. In the BHK-21, Hela, and Vero cells, the rVTT-TA35-TJ infected cells formed detectable plaques, while the other two
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types of cells did not. However, the number of plaques formed was significantly less than in VTT-infected cells. These results indicate that the deletion of the two gene fragments
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significantly reduces the virulence of the vaccinia virus Tian Tan strain, thereby increasing its safety as a vaccine vector. In the in vivo experiments, it was found that the high-dose VTT group reached the lowest
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weight at day 9 post infection, with an average reduction of 23%. The body weight change in the middle-dose VTT group was similar to the high dose group. In contrast, the body weights of the mice in the high, middle, and low dose groups of rVTT-TA35-TJ were not significantly different than the PBS group, indicating that the virulence of the vaccinia virus was greatly reduced after knocking out the two gene fragments. Similarly, it has been reported that inoculation of MVA did not result in a decrease in the weight of the inoculated
mice compared to mice inoculated with the wild-type virus (Melamed et al., 2013). In the skin lesion formation experiment in rabbits, the pocks formed by high-dose VTT infection peaked on day 3 post inoculation, followed by a slow downtrend. In contrast, the rVTT-TA35-TJ group incurred only mild swelling, and the low-dose group recovered on day 13, consistent with the results of previous assays. In addition, the vaccinia virus NYVAC strain was obtained by continuously knocking out
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18 non-essential genes, such as virulence and host-wide genes of the Copenhagen strain. Compared to the wild strain, it has the advantages of low neurotoxicity in suckling mice and
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weak pathogenicity in nude mice (Tartaglia et al., 1992). rVTT-TA35-TJ also displays high
safety in terms of neurotoxicity. After the intracranial injection of the viruses the wild-type group all died on day 8 post infection, while the mice in the rVTT-TA35-TJ high-, medium-,
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and low-dose group survived until the end of the experiment, and with only mild neurological
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symptoms during the first 2 days post inoculation.
Cytokines play an important role in the body’s immune response, such as antiviral and
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mediated inflammatory responses. Research has shown that IL-4, IL-10, and IFN-γ play an
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important role in the immune response of mice to VV infection (van Den Broek et al., 2000).
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Most studies on the deletion of immunoregulatory genes in VV have shown that the absence of several VV genes reduces the toxicity of the virus (Smith et al., 2013), but the effects on immunogenicity is variable. For example, deletion of genes, such as N2L and C16L, was
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found to have no effect on immunogenicity (Alcami and Smith, 1995; Fahy et al., 2008; Ferguson et al., 2013). However, deletion of these immunomodulatory genes (E3L,
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B15R/B16R, A41L, B22R, C12L, and C6L) from different strains (mainly WR and MVA) was found to increase the immunogenicity of this virus. Here, it was found that the recombinant vaccinia virus rVTT-TA35-TJ, lacking the TJ and TA35R gene fragments, had
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no significant difference in immunogenicity level compared to the parental VTT strain. In summary, knocking out the TJ and TA35R gene fragments not only simplified the construction process of the recombinant vaccinia virus, also increased safety compared to VTT, while maintaining its immunogenicity as a vaccine. Therefore, rVTT-TA35-TJ has potential as a new virus vector and vaccine for the prevention or treatment of diseases caused by different pathogens.
Conflicts of interest statement The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Author contributions Conceived and designed the experiments: YQL, XL, LLS and NYJ. Performed the experiments: YQL, SC, JBF, YLZ, BB, WJL, XZY, JW and NYJ. Analyzed the data: YQL, XL, LLS and NYJ. Contributed reagents/materials/analysis tools: JBF, YLZ, XZY, XL and JCH. Wrote the paper: YQL and NYJ. All authors read and approved the final manuscript.
Acknowledgments
This work was supported by the National Science and Technology Major Project (Major New
Key
Research
and
Development
Program
of
China
[grant
number
N
National
U
Drugs Innovation and Development) [grant number 2018ZX09301053-004-001], the
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2016YFC1200900] and the Major Technological Program of Changchun City [grant number
M
16ss11].
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REFERENCES
Adelfinger, M., Gentschev, I., Grimm de Guibert, J., Weibel, S., Langbein-Laugwitz, J., Hartl, B., Murua Escobar, H., Nolte, I., Chen, N.G., Aguilar, R.J., Yu, Y.A., Zhang, Q.,
PT
Frentzen, A., Szalay, A.A., 2014. Evaluation of a new recombinant oncolytic vaccinia virus strain GLV-5b451 for feline mammary carcinoma therapy. PloS one 9(8),
CC E
e104337.
Alcami, A., Smith, G.L., 1995. Cytokine receptors encoded by poxviruses: a lesson in cytokine biology. Immunology today 16(10), 474-478.
A
Bray, M., Wright, M.E., 2003. Progressive vaccinia. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 36(6), 766-774.
Casey, C.G., Iskander, J.K., Roper, M.H., Mast, E.E., Wen, X.J., Torok, T.J., Chapman, L.E., Swerdlow, D.L., Morgan, J., Heffelfinger, J.D., Vitek, C., Reef, S.E., Hasbrouck, L.M., Damon, I., Neff, L., Vellozzi, C., McCauley, M., Strikas, R.A., Mootrey, G., 2005. Adverse events associated with smallpox vaccination in the United States,
January-October 2003. Jama 294(21), 2734-2743. Cono, J., Casey, C.G., Bell, D.M., Centers for Disease, C., Prevention, 2003. Smallpox vaccination
and
Recommendations
adverse
reactions.
and reports :
Guidance
for
clinicians.
MMWR.
Morbidity and mortality weekly report.
Recommendations and reports 52(RR-4), 1-28. Eckart, R.E., Love, S.S., Atwood, J.E., Arness, M.K., Cassimatis, D.C., Campbell, C.L.,
IP T
Boyd, S.Y., Murphy, J.G., Swerdlow, D.L., Collins, L.C., Riddle, J.R., Tornberg, D.N.,
Grabenstein, J.D., Engler, R.J., Department of Defense Smallpox Vaccination Clinical
SC R
Evaluation, T., 2004. Incidence and follow-up of inflammatory cardiac complications after smallpox vaccination. Journal of the American College of Cardiology 44(1), 201-205.
U
Fahy, A.S., Clark, R.H., Glyde, E.F., Smith, G.L., 2008. Vaccinia virus protein C16 acts
A
general virology 89(Pt 10), 2377-2387.
N
intracellularly to modulate the host response and promote virulence. The Journal of
Ferguson, B.J., Benfield, C.T., Ren, H., Lee, V.H., Frazer, G.L., Strnadova, P., Sumner, R.P.,
M
Smith, G.L., 2013. Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes
ED
virulence. The Journal of general virology 94(Pt 9), 2070-2081. Garcia-Arriaza, J., Arnaez, P., Gomez, C.E., Sorzano, C.O., Esteban, M., 2013. Improving Adaptive and Memory Immune Responses of an HIV/AIDS Vaccine Candidate
PT
MVA-B by Deletion of Vaccinia Virus Genes (C6L and K7R) Blocking Interferon Signaling Pathways. PloS one 8(6), e66894.
CC E
Jacobs, B.L., Langland, J.O., Kibler, K.V., Denzler, K.L., White, S.D., Holechek, S.A., Wong, S., Huynh, T., Baskin, C.R., 2009. Vaccinia virus vaccines: past, present and future. Antiviral research 84(1), 1-13.
A
Kan, S., Wang, Y., Sun, L., Jia, P., Qi, Y., Su, J., Liu, L., Yang, G., Liu, L., Wang, Z., Wang, J., Liu, G., Jin, N., Li, X., Ding, Z., 2012. Attenuation of vaccinia Tian Tan strain by removal of viral TC7L-TK2L and TA35R genes. PloS one 7(2), e31979. Kemper, A.R., Davis, M.M., Freed, G.L., 2002. Expected adverse events in a mass smallpox vaccination campaign. Effective clinical practice : ECP 5(2), 84-90. Lane, J.M., Goldstein, J., 2003. Adverse events occurring after smallpox vaccination.
Seminars in pediatric infectious diseases 14(3), 189-195. Lederman, E., Miramontes, R., Openshaw, J., Olson, V.A., Karem, K.L., Marcinak, J., Panares, R., Staggs, W., Allen, D., Weber, S.G., Vora, S., Gerber, S.I., Hughes, C.M., Regnery, R., Collins, L., Diaz, P.S., Reynolds, M.G., Damon, I., 2009. Eczema vaccinatum resulting from the transmission of vaccinia virus from a smallpox vaccinee: an investigation of potential fomites in the home environment. Vaccine
IP T
27(3), 375-377.
Li, X., Jin, N., Mi, Z., Lian, H., Sun, L., Li, X., Zheng, H., 2006. Antitumor effects of a
SC R
recombinant fowlpox virus expressing Apoptin in vivo and in vitro. International journal of cancer 119(12), 2948-2957.
Liu, J., McFadden, G., 2015. SAMD9 is an innate antiviral host factor with stress response
U
properties that can be antagonized by poxviruses. Journal of virology 89(3),
N
1925-1931.
A
Mackett, M., Yilma, T., Rose, J.K., Moss, B., 1985. Vaccinia virus recombinants: expression of VSV genes and protective immunization of mice and cattle. Science 227(4685),
M
433-435.
ED
MacNeil, A., Reynolds, M.G., Damon, I.K., 2009. Risks associated with vaccinia virus in the laboratory. Virology 385(1), 1-4.
Melamed, S., Wyatt, L.S., Kastenmayer, R.J., Moss, B., 2013. Attenuation and of
PT
immunogenicity
host-range
extended
modified
vaccinia
virus
Ankara
recombinants. Vaccine 31(41), 4569-4577.
CC E
Meulemans, G., Letellier, C., Gonze, M., Carlier, M.C., Burny, A., 1988. Newcastle disease virus F glycoprotein expressed from a recombinant vaccinia virus vector protects
A
chickens against live-virus challenge. Avian pathology : journal of the W.V.P.A 17(4), 821-827.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immunological methods 65(1-2), 55-63. Noisumdaeng, P., Pooruk, P., Kongchanagul, A., Assanasen, S., Kitphati, R., Auewarakul, P., Puthavathana, P., 2013. Biological properties of H5 hemagglutinin expressed by
vaccinia virus vector and its immunological reactivity with human sera. Viral immunology 26(1), 49-59. Pastoret, P.P., Brochier, B., 1996. The development and use of a vaccinia-rabies recombinant oral vaccine for the control of wildlife rabies; a link between Jenner and Pasteur. Epidemiology and infection 116(3), 235-240. Qin, L., Favis, N., Famulski, J., Evans, D.H., 2015. Evolution of and evolutionary
IP T
relationships between extant vaccinia virus strains. Journal of virology 89(3), 1809-1824.
SC R
Reed LJ, M., H, 1938. A simple method of estimating fifty percent endpoints. Am J Hyg 27, 493–497.
Rehm, K.E., Roper, R.L., 2011. Deletion of the A35 gene from Modified Vaccinia Virus
U
Ankara increases immunogenicity and isotype switching. Vaccine 29(17), 3276-3283.
N
Remy-Ziller, C., Germain, C., Spindler, A., Hoffmann, C., Silvestre, N., Rooke, R.,
A
Bonnefoy, J.Y., Preville, X., 2014. Immunological characterization of a modified vaccinia virus Ankara vector expressing the human papillomavirus 16 E1 protein.
M
Clinical and vaccine immunology : CVI 21(2), 147-155.
ED
Rintoul, J.L., Wang, J., Gammon, D.B., van Buuren, N.J., Garson, K., Jardine, K., Barry, M., Evans, D.H., Bell, J.C., 2011. A selectable and excisable marker system for the rapid creation of recombinant poxviruses. PloS one 6(9), e24643.
PT
Roper, R.L., 2006. Characterization of the vaccinia virus A35R protein and its role in virulence. Journal of virology 80(1), 306-313.
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Smith, G.L., Benfield, C.T., Maluquer de Motes, C., Mazzon, M., Ember, S.W., Ferguson, B.J., Sumner, R.P., 2013. Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity. The Journal of general virology 94(Pt 11), 2367-2392.
A
Tartaglia, J., Perkus, M.E., Taylor, J., Norton, E.K., Audonnet, J.C., Cox, W.I., Davis, S.W., van der Hoeven, J., Meignier, B., Riviere, M., et al., 1992. NYVAC: a highly attenuated strain of vaccinia virus. Virology 188(1), 217-232. van Den Broek, M., Bachmann, M.F., Kohler, G., Barner, M., Escher, R., Zinkernagel, R., Kopf, M., 2000. IL-4 and IL-10 antagonize IL-12-mediated protection against acute vaccinia virus infection with a limited role of IFN-gamma and nitric oxide synthetase
2. Journal of immunology 164(1), 371-378. Wang, Y., Kan, S., Du, S., Qi, Y., Wang, J., Liu, L., Ji, H., He, D., Wu, N., Li, C., Chi, B., Li, X., Jin, N., 2012. Characterization of an attenuated TE3L-deficient vaccinia virus Tian Tan strain. Antiviral research 96(3), 324-332. Wollenberg, A., Engler, R., 2004. Smallpox, vaccination and adverse reactions to smallpox vaccine. Current opinion in allergy and clinical immunology 4(4), 271-275.
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Yu, W., Fang, Q., Zhu, W., Wang, H., Tien, P., Zhang, L., Chen, Z., 2010. One time intranasal
vaccination with a modified vaccinia Tiantan strain MVTT(ZCI) protects animals
SC R
against pathogenic viral challenge. Vaccine 28(9), 2088-2096.
Zhu, W., Fang, Q., Zhuang, K., Wang, H., Yu, W., Zhou, J., Liu, L., Tien, P., Zhang, L., Chen, Z., 2007. The attenuation of vaccinia Tian Tan strain by the removal of the viral
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A
N
U
M1L-K2L genes. Journal of virological methods 144(1-2), 17-26.
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Figure Legends:
Fig. 1. Construction of an attenuated vaccinia virus, rVTT-TA35-TJ. Structure of the plasmid vector (A), recombinant vaccinia virus (B), and gene-deleted virus
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(C). EGFP was inserted as a screening marker at the TJ2R gene site of VTT (A) and driven by the synthetic early/late promoter (pE/L) to obtain the recombinant virus with deletion of
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the TJ2R gene, but containing the EGFP gene (B). The recombinant virus rVTT-J was generated through Cre/loxP site-specific recombination to remove the EGFP gene (C). The same
method
was
used
to
successively
knock
out
TA35R.
Ultimately,
the
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two-segment-deleted attenuated strain rVTT-TA35-TJ was obtained. T, termination signal of vaccinia virus; loxP, loxP site; Cre, Cre recombinase.
Fig. 2. Screening and identification of mutants. The fused EGFP was expressed by the mutants rVTT-J-EGFP (A1) and rVTT-A35-J-EGFP (B1) in BHK-21 cells. The virus-infected cells were identified by visualization of isolated
fluorescent plaques in the same visual fields. Non-fluorescent plaques were observed for rVTT-J(A2) and rVTT-TA35-TJ (B2) mutants in BHK-21 cells (magnification 200×, A–B); PCR was performed to identify the final mutant rVTT-TA35-TJ (C-F). (C) Lane 2: positive control containing the partial segments TA35R gene (307 bp), lane 4: positive control containing the partial segments TJ2R gene (261 bp), and lanes 1 and 3: PCR products of the rVTT-TA35-TJ genome; (D) Lane 1: positive control containing the TA35R gene (751 bp),
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Lane 2: PCR products of the rVTT-TA35-TJ genome; (E) Lane 1: positive control containing the TJ2R gene (551 bp), Lane 2: PCR products of the rVTT-TA35-TJ genome; Evaluation of
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the genetic stability of the recombinant virus by PCR. (D) TA35R gene of rVTT-TA35-TJ (lanes 2–5) and (E) TJ2R gene of rVTT-TA35-TJ (lanes 2–5). PCR of all gene-deleted mutants produced negative results, compared to the corresponding positive PCR controls
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(lane 1).
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Fig. 3. In vitro replication of rVTT-TA35-TJ and VTT.
Growth curve of rVTT-TA35-TJ and VTT in BHK-21 (A), MDCK (B), Hela (C), Vero (D),
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and PK-15 (E) cells. Cells were infected with rVTT-TA35-TJ and VTT at an MOI of 0.5, and
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the virus titers were measured at 3, 12, 24, and 48 h post infection.
Fig. 4. In vitro cytotoxicity of rVTT-TA35-TJ and VTT.
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BHK-21 (A), MDCK (B), Hela (C), Vero (D), and PK-15 (E) cells were seeded in 96-well plates (1 × 104 cells/well) and infected with rVTT-TA35-TJ and VTT at an MOI of 0.5. Cell
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viability was measured every 24 h over a 96 h period. All measurements were performed in triplicate. Data are presented as the mean ± standard deviation. Confluent monolayers of BHK-21, MDCK, Hela, Vero, and PK-15 cells in 12-well plates were infected with 0.5 MOI
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of rVTT-TA35-TJ and VTT, then stained with 0.4% crystal violet after plaque phenotypes formed. The pathogenicity of rVTT-TA35-TJ decreased in all five cell lines (compared with VTT as the control) (F).
Fig. 5. Virulence of rVTT-TA35-TJ or VTT in rabbits. Rabbits were infected with 105, 106, or 107 PFU of rVTT-TA35-TJ or VTT (as a positive
control) or PBS (as a negative control) by intradermal injection. Infection with 10 5, 106, and 107 PFU of rVTT-TA35-TJ resulted in mild swelling by day 2, and no visible scars were observed by day 10 at the sites of infection (A). However, infection with VTT resulted in the formation of severe lesions at the infection site, and the appearance of ulcerations. The size of the lesions increased until they reached a maximum at day 3, after which they gradually decreased, but left scars (A). The diameter of the lesions was associated with the VTT and
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rVTT-TA35-TJ dose (B).
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Fig. 6. Virulence of VTT and rVTT-TA35-TJ in mice after intranasal and intracranial infection.
(A) Body weights were monitored in mice that were intranasally infected with 105, 106, or
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107 PFU of rVTT-TA35-TJ or VTT (as a positive control) or PBS (as a negative control).
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Error bars indicate the standard error of the mean, and differences between groups were
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determined by two-way repeated measures analysis of variance. Mice inoculated with VTT showed significant signs of illness, and a clear positive correlation was found between the
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viral dose and weight loss (P < 0.05). No distinct weight changes or signs of illness were observed in the animals inoculated with rVTT-TA35-TJ or PBS.
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(B) Mice were inoculated intracranially with 105, 106, or 107 PFU of rVTT-TA35-TJ or VTT (PBS in the negative control group), and the survival rates were observed for 14 days. All the
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mice inoculated with rVTT-TA35-TJ survived, while all the mice infected with VTT died.
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Fig. 7. Immune responses to rVTT-TA35-TJ or VTT in vaccinated mice. BALB/c mice were inoculated intramuscularly with 106 PFU rVTT-TA35-TJ or VTT, and given a booster immunization of the same dose in 0.1 ml PBS three weeks later. The serum
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samples were harvested at weeks 3 and 5 post immunization. The IL-2 (A), IL-4 (B), IL-10 (C), and IFN-γ (D) levels in the two vaccinated groups were measured by mouse ELISA kits. All measurements were performed in triplicate. Data are presented as the mean ± SD values. VTT-specific IgG levels were measured by ELISA on VTT-coated plates and then read by a microplate reader. Data are presented as mean ± SD (E). The neutralization antibody titer was calculated by determining the highest serum dilution required to generate a 50% viral plaque
reduction (F). All measurements were performed in triplicate. Data are presented as the mean
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± SD values.
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S0168-1702(18)30306-X https://doi.org/10.1016/j.virusres.2018.06.017 VIRUS 97437
To appear in:
Virus Research
Received date: Revised date: Accepted date:
18-5-2018 29-6-2018 30-6-2018
Please cite this article as: Li Y, Chen S, Fang J, Zhu Y, Bai B, Li W, Yin X, Wang J, Liu X, Han J, Li X, Sun L, Jin N, Construction of an Attenuated Tian Tan Vaccinia Virus Strain by Deletion of TA35R and TJ2R Genes, Virus Research (2018), https://doi.org/10.1016/j.virusres.2018.06.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Construction of an Attenuated Tian Tan Vaccinia Virus Strain by Deletion of TA35R and TJ2R Genes
Yiquan Li a,b, Shuang Chen a,b,c, Jinbo Fang b,d, Yilong Zhu b,d, Bing Bai b,d, Wenjie Li b,d,e,
a,b,d,f,h*
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Xunzhe Yin b,d, Jing Wang b, Xing Liu b, Jicheng Han a,b, Xiao Li b,d,f*, Lili Sun b,g*, Ningyi Jin
Medical College, Yanbian University, Yanji 133002, P. R. China
b
Institute of Military Veterinary Medicine, Academy of Military Medical Science, Changchun
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a
130122, P. R. China
School of Medical Inspection, Jilin Medical University, Jilin 132013, P. R. China
d
Changchun University of Chinese Medicine, Changchun 130021, P. R. China
e
College of Animal Science and Technology, Guangxi University, Nanning 530005, P. R.
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c
f
A
China
Institute of Virology, Wenzhou University Town, Wenzhou 325035, P. R. China Department of Head and Neck Surgery, Tumor Hospital of Jilin Province, Changchun
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g
h
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130012, P. R. China
Jiang su Co-innovation Center for Prevention and Control of Important Animal Infectious
Diseases and Zoonoses, Yangzhou 225009, P. R. China
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*Corresponding author
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Corresponding author: Ningyi Jin, Xiao Li and Lili Sun E-mail: [email protected] (JN); [email protected] (LX); [email protected] (SL)
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Tel: +86-0431-86985923 Fax: +86-0431-87985861 Address: Liuying west road, 666, Jingyue economic & technological development zone, Changchun, Jilin, 130122, P.R.China
Highlights
We construct an attenuated multi-genes deficient vaccinia virus Tian Tan strain.
Replication ability of the attenuated virus is not reduced.
Cell toxicity of the attenuated virus is significantly decreased.
The in vivo virulence of the attenuated virus is reduced.
The attenuated virus is a candidate vector with increased safety.
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Abstract
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rVTT-TA35-TJ, an attenuated vaccinia virus Tian Tan strain (VTT), was constructed by
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knocking out two non-essential gene fragments (TA35R and TJ2R) related to virulence, immunomodulation, and host range; and by combining double marker screening with
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exogenous and endogenous selectable marker knock-out techniques. Here, the shuttle plasmids pSK-TA35 and pSK-TJ were constructed, containing two pairs of recombinant
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arms: early and late strong promoter pE/L and EGFP as an exogenous selectable marker. The recombinant vaccinia virus rVTT-TA35-TJ without exogenous selection markers was then
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obtained through homologous recombination technology and the Cre/loxP system. Knocking out the two gene fragments does not affect the replication ability of the virus and displays a good genetic stability. Furthermore, a series of in vivo and in vitro experiments demonstrate
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that although virulence of rVTT-TA35-TJ is attenuated significantly, high immunogenicity was maintained. These results support the potential development of rVTT-TA35-TJ as a safe viral vector or vaccine. Keywords: Attenuated vaccinia virus vector; Tian Tan strain; virulence; immunogenicity
1. Introduction
The vacinia virus (VV) is a member of the genus Orthopoxvirus, of the family Poxviridae. The vaccinia virus has a genome size of 185 to 200 kb, divided into central conservative areas and flanking areas, and encodes 200 proteins. The highly conserved central part of the genome, which comprises the majority of the VV genome, contains the essential genes that play a critical role in virus replication, such as DNA replication, transcription, and viral particle assembly (Qin et al., 2015). The genes present at both ends of
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the genome are commonly used to identify species or host specificities, and to encode
proteins that regulate the host immune system and virulence factors (Liu and McFadden,
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2015).
Vaccinia virus Tian Tan strain (VTT), a unique vaccinia virus strain in China, has played a key role in the eradication of smallpox in China, but the virus also causes side effects, such
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as fever, fatigue, ocular vaccinia infection, gangrenosa vaccinia, and encephalitis (Lederman
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et al., 2009; Wollenberg and Engler, 2004). These can be severe or even life-threatening,
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especially in patients with immunosuppression, eczema, chronic skin diseases, and heart disease (Bray and Wright, 2003; Casey et al., 2005; Eckart et al., 2004; Kemper et al., 2002).
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Therefore, the safety of vaccinia virus needs further developed.
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As a double-stranded DNA virus, vaccinia virus has unique biological characteristics. First, unlike other DNA viruses, vaccinia virus is present in the cytoplasm during the entire infection cycle from virus entry to progeny virus formation. Therefore, the virus's DNA does
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not integrate into the host's genome. Second, vaccinia virus has a large genome and can accommodate at least 25 kb of foreign genes. In addition, vaccinia virus particles are
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relatively stable and can be stored as dry powder form, making them easy to transport for clinical application. It is precisely due to the above advantages of vaccinia virus that many scientists have tried to use vaccinia virus as a carrier to express antigenic proteins of various
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diseases, and have conducted various vaccine research. Many vaccines have been developed with vaccinia virus as a carrier to prevent other
diseases (Adelfinger et al., 2014; Garcia-Arriaza et al., 2013; Noisumdaeng et al., 2013; Remy-Ziller et al., 2014), such as hepatitis A virus, hepatitis B virus, herpes simplex virus, and human immunodeficiency virus. For example, the genetically engineered vaccine against the rabies virus glycoprotein is synthesized using the VV Copenhagen strain as the vector
(Pastoret and Brochier, 1996), the human cytomegalovirus pp65 protein uses the ALVAC vector, the vesicular stomatitis virus glycoprotein protein uses the VV Western Reserve (WR) strain as the vector (Mackett et al., 1985), and the Newcastle Disease Virus fusion protein uses the VV Elstree strain as the vector (Meulemans et al., 1988). Although significant progress has been made in the study of VV as a vector, there are still some limitations, such as the selection of vaccinia viruses after viral recombination, the insertion and deletion of
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exogenous selection markers, and the complexity of vector construction procedures (MacNeil et al., 2009). Currently, reducing the side effects of VV vaccines, simplifying the preparation
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process, and improving the efficiency of vector vaccines are research hotspots of VV vectors.
Here, we obtained the attenuated strain rVTT-TA35-TJ by knocking out the following two non-essential gene segments of the VTT strain: TA35R (138,881139,570) and TJ2R
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(80,294-80,827). The knockout fragments TA35R contain virulence-related genes and
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immunomodulation-related genes. NYVAC, as one of the most successful gene-knockout
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attenuated VV vectors, was obtained by phenotypic attenuation after knockout of the 18 ORFs, containing the TA35R gene that is a highly expressed gene responsible for the
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formation of A-type inclusion bodies (Tartaglia et al., 1992). The production of the thymidine
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kinase (TK) gene of vaccinia virus can be inhibited by using deoxyuridine-like (Brdu) to block the synthesis of the viral nucleic acid, inhibiting the replication of vaccinia virus in the cell, allowing for selection of the vaccinia virus lacking the TK gene. The TK of VTT is
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encoded by the TJ2R gene segment in the genome which can be used as an endogenous selection marker for the screening of recombinant vaccinia virus.
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To further simplify the procedure of recombinant vaccinia virus construction, the
screening process was optimized: a double labeling screening method using both exogenous selection marker enhanced green fluorescent protein (EGFP) and an endogenous selection
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marker TK (TJ2R), and knockout of the exogenous selection marker was achieved by introducing the Cre/LoxP system into the shuttle vector plasmid (Rintoul et al., 2011). These strategies significantly shorten the cycle of recombinant vaccinia virus and improve the efficiency of screening for recombinants. Subsequent in vitro and in vivo experiments in mice and rabbits further examined the recombinant vaccinia virus lacking TA35R and TJ2R from vaccinia Tian Tan strain in relation to its virulence and immunogenicity.
2. Materials and Methods 2.1 Cells, viruses, and animals BHK-21, MDCK, Hela, Vero, and PK-15 were purchased from the China Center for Type Culture Collection (CCTCC). These cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Hyclone, Beijing, China) with 10% fetal bovine serum (FBS) (Hyclone, Beijing, China) and penicillin (10,000 U/mL)/1% streptomycin (10 mg/mL) (Hyclone,
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Beijing, China). The VTT strain (GenBank accession no. AF095689) was obtained from the Institute of Virology at the Chinese Center for Disease Control and Prevention.
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Three-week-old and six-week-old BALB/c male mice and New Zealand male white rabbits were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences of China.
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The animal experimental protocols were approved by the Institutional Animal Care and
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Use Committee (IACUC) of the Chinese Academy of Military Medical Science, Changchun,
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China (10ZDGG007). All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
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2.2 Construction of VTT shuttle plasmid
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To construct the genes deleted VTT, TA35 (138,881139,570) and TJ (80,294-80,827) were selected as deletions based on results of whole-genome sequence analysis of the VTT strain (unpublished data). The shuttle plasmid pSK-TA35 containing TA35 gene recombinant
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left arm (138,353 – 138,881 bp)-LoxP-PE/L-EGFP-T5NT-LoxP, TA35 gene recombinant right arm (139,570 – 140,607 bp), and shuttle plasmid pSK-TJ containing TJ gene left
arm
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recombinant
(79,784–80,294
bp)-Loxp-PE/L-EGFP-T5NT-Loxp,
TJ
gene
recombinant right arm (80,827–81,855 bp) sites were synthesized from Shanghai Generay Biotech Co., Ltd of China.
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2.3 Construction and genetic stability analysis of gene deletion VTT The recombinant virus rVTT-A35-J-EGFP, in which the TA35 and TJ genes were
replaced with EGFP, was constructed by homologous recombination. rVTT-TA35-TJ, lacking the TA35 and TJ genes, was obtained from rVTT-A35-J-EGFP, following the knockout of EGFP by Cre/Loxp system. BHK-21 cells were infected with VTT at a MOI of 0.1 for 2 h. Then, the cells were transfected with the mixture of the shuttle pSK-TJ and Effectene
Transfection Reagent (QIAGEN). Following culture for 72 h, the recombinant virus was released by three freeze-thaw cycles and used for further infection. Under an inverted fluorescence microscope, green fluorescent plaques were picked out, and this purification process was repeated six times until the monoclonal fluorescent plaque (i.e., the recombinant virus rVTT-J-EGFP) was purified. rVTT-J-EGFP and the plasmid pVAX-Cre were co-transfected into BHK-21 cells, and rVTT-J lacking the TJ gene was isolated by screening
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the non-green fluorescent plaques (i.e., the non-EGFP-expressing virus) under a fluorescence microscope; this step was also repeated six times. The TA35 gene was knocked out in the
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same manner. Ultimately, both the TA35 and TJ genes were deleted, and the vector was named rVTT-TA35-TJ.
rVTT-TA35-TJ was identified by PCR. The genome of rVTT-TA35-TJ was extracted to
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use as template for amplifying the partial segments TA35 (307 bp) and TJ (261 bp) by PCR.
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The primers are as follows: TA35-1F; CAGCGTGATTCTTACCAGATATT, TA35-1R;
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TGTTGCGAGCATTACTGCGTTTA, TJ-1F; GATTGGAAGCAACTAAACTATGT, TJ-1R; CCTCCTATTATTTCTATCTCGGT. At the same time, we also designed primers on both ends
follows:
TA35-2F;
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The primers are as
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of the recombination arm and amplified fragments TA35 (751 bp) and TJ (551 bp) by PCR.
ACTGGACGGCGTCCATT,
TJ-2F;
AATGTCCAGGATTCTCCTA, TA35-2R; AGTGAACAATAATTAATTC,
TJ-2R;
AATTTAATCAATGTTTAT. The PCR reaction conditions were 95°C 5 min, 30 cycles of
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95°C 30 s, 57°C 30 s, 72°C 30 s, and a final extension at 72°C for 10 min. The genome of VTT was used as a control.
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The genomes of the 5th, 10th, 15th, and 20th passage in rVTT-TA35-TJ-infected
BHK-21 cells were extracted to detect genetic stability of rVTT-TA35-TJ by PCR. The genome of VTT was used as a control.
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2.4 Characterization of rVTT-TA35-TJ The characterization of rVTT-TA35-TJ was performed by growth curve, plaque
formation assay, and MTS assay. To detect and observe the formation of virus plaques of rVTT-TA35-TJ-infected cells, the BHK-21, MDCK, Hela, Vero, and PK-15 cells were infected with rVTT-TA35-TJ and VTT at 0.5 MOI for 48 h. Then, the cytopathic effects of these infected cells were observed with a fluorescence microscope.
To study the multiplication of rVTT-TA35-TJ, BHK-21, MDCK, Hela, Vero, and PK-15 cells were infected with rVTT-TA35-TJ and VTT at 0.5 MOI. Then, the viral titer at 3, 12, 24, and 48 h was determined. BHK-21 cell was cultured in 24-well cell culture plates for 24 h. A serial 8-fold serial dilution of the rVTT-TA35-TJ and VTT was performed. The cell culture medium was discarded, and then the virus was inoculated at a virus dose of 200 ul/well, with 4 repeat wells for each dilution. Incubate at 37 °C, 5% CO2 for 5 days. Calculation formula:
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PFU = (average number of virus plaques × dilution) / inoculum volume. The growth curves were then extrapolated from the viral titers at each time point.
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To assess the virulence of rVTT-TA35-TJ, BHK-21, MDCK, Hela, Vero, and PK-15 cells were infected with rVTT-TA35-TJ and VTT at 0.5 MOI and then cultured at 37°C with 5% CO2. The virus-infected cells were cultured for 2 h following the addition of 20 μl of MTS
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solution (Promega) at 24, 48, 72, and 96 h post infection. The number of viable cells was
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quantified by measuring optical density (OD) at 490 nm. Cell viability was calculated
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according to the following formula: 100 × (absorbance of culture in the treated
2.5 Safety analysis of rVTT-TA35-TJ
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wells/absorbance of culture in the control wells) (Li et al., 2006; Mosmann, 1983).
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The safety of rVTT-TA35-TJ was evaluated by detecting neurotoxicity, measuring weight changes in mice, and measuring pock size in New Zealand white rabbits. Six-week-old male BALB/c mice were divided into 7 groups (n = 10). Group 1 received 1×105 PFU of
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rVTT-TA35-TJ in 20 μl of normal saline by intranasal inoculation, group 2 received 1×10 6 PFU of rVTT-TA35-TJ, group 3 received 1×107 PFU of rVTT-TA35-TJ, group 4 received
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1×105 PFU of VTT, group 5 received 1×106 PFU of VTT, group 6 received 1×107 PFU of VTT, and group 7 received 20 μl of normal saline. The body weight of each mouse was recorded every day for 25 days post inoculation.
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Three-week-old male BALB/c mice were divided into 7 groups (n = 10) and inoculated intracranially with 10 μl of rVTT-TA35-TJ and VTT diluted with normal saline at doses of 1×105 PFU/10 μl PBS, 1×106 PFU/10 μl PBS, or 1×107 PFU/10 μl. Grouping arrangement was identical as above; the control group was inoculated with PBS. Deaths were observed for 14 d post inoculation. The intracranial 50% lethal infectious dose (ICLD50) was calculated using the Reed–Muench method (Reed LJ, 1938).
The backs of male New Zealand white rabbits received 1×105 PFU/100 μl of rVTT-TA35-TJ and VTT, 1×106 PFU/100 μl of rVTT-TA35-TJ and VTT, or 1×107 PFU/100 μl of rVTT-TA35-TJ and VTT by intradermal injection. Normal saline was used as a control group. Every concentration was injected into two rabbits and repeated three times. The pock size was measured by Electronic vernier caliper daily for 18 d post injection. 2.6 Immunogenicity of rVTT-TA35-TJ in mice
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The immunogenicity was estimated by detecting the levels of VTT-specific antibody, IL-2, IL-4, IL-10, IFN-γ, and neutralizing antibody titer. Six-week-old BALB/c mice (n = 10)
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were inoculated intramuscularly with 1×106 PFU/ml normal saline with rVTT-TA35-TJ and VTT; the control group of goats was inoculated with normal saline. The sera separated from the weekly blood samples were used for the detection of IL-2, IL-4, IL-10, and IFN-γ with
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ELISA kits (MYBioSource) after vaccinating with rVTT-TA35-TJ. The levels of
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VTT-specific IgG were measured by ELISA. The purified VTT was diluted to 1×106 PFU/ml. The virus was diluted 10 times with the coating solution, and then added to the ELISA plate
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at 100 μL/well, and stay overnight at 4℃. Discard the coating solution, wash the plate 3
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times, and Add 200 μL blocking solution per well and incubate at 37°C for 2 h. Wash the plate 5 times and Add 100 μL of 10-fold diluted serum sample to each well and incubate for 2
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h at 37°C. Wash the plate 5 times and Add 100 μL of HRP-labeled antibody to each well and incubate for 2 h at 37°C. Wash the plate 5 times and Add 100 μL 1×TMB solution to each
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well and incubate at room temperature for 15 min. Add 50 μL of stop solution per well. And the optical density (OD) was detected at 450 nm. The neutralization assay was performed as
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described previously (Kan et al., 2012). The results were calculated using the method of Reed and Muench (Reed LJ, 1938). 2.7 Statistical analysis
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The statistical analysis was performed using data from at least three independent
experiments using the Statistical Package for the Social Sciences (SPSS) statistical software package (version 15.0; SPSS, Inc., Chicago, IL, USA), and the results were obtained using GraphPad Prism version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA). Student’s t-tests were performed for all analyses and differences among groups were determined by one-way ANOVA with Tukey's post hoc test.
P < 0.05 was considered to indicate statistical
significance. Data are presented as the mean ± standard deviation (SD).
3. Results 3.1 Construction and identification of rVTT-TA35-TJ The shuttle plasmids pSK-TA35 and pSK-TJ were identified by double-restriction enzyme digestion with EcoRI and PstI. The EGFP fragment (720 bp) was obtained by double
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digestion of the shuttle plasmids. These results indicate that the two shuttle plasmids were constructed successfully.
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The screening process and PCR results for rVTT-TA35-TJ are shown in Figure 2A-E. From Figure 2C we can see that the 261 bp and 307 bp fragments cannot be amplified from
the rVTT-TA35-TJ genome, but can be amplified from the VTT genome; and from Figure
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2D-E we can see that the fragments TA35 (751 bp) and TJ (551 bp) can be amplified from the
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VTT genome, but cannot be amplified from the rVTT-TA35-TJ genome demonstrating that
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the partial segment of TJ and TA35 genes was knocked out.
rVTT-TA35-TJ was continuously passaged 20 times, and the PCR products of TA35 (307
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bp) or TJ (261 bp) of the genome from the 5th, 10th, 15th, and 20th passage in VTT-infected
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BHK-21 cells could be amplified. However, PCR amplification of the 5th, 10th, 15th, and 20th generation of the rVTT-TA35-TJ genome under the same conditions did not produce the corresponding bands. These results demonstrate that rVTT-TA35-TJ has good genetic
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stability (Fig. 2F-G).
3.2 Characterization of rVTT-TA35-TJ
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The growth curve results of rVTT-TA35-TJ and VTT are shown in Figure 3. The two
viruses have similar growth curves in the same cells and no significant difference in titers of rVTT-TA35-TJ with VTT was observed, indicating that the deletion of TA35 and TJ gene
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reduce viral titer without affecting normal replication of virus. The cytotoxicity of rVTT-TA35-TJ and VTT was measured by MTS, and the results are
shown in Figure 4A-D. The number of living cells decreased with time, and in the BHK, Vero, and PK-15 cells, the survival rate of VTT-infected cells was significantly lower than the rVTT-TA35-TJ-infected cells at 48 and 72 h (P < 0.01). At 96 h, the survival rate of VTT-infected cells was significantly lower than the rVTT-TA35-TJ-infected cells. These
results suggest that the deletion of TA35 and TJ gene in rVTT-TA35-TJ significantly reduced the in vitro virulence of VTT. Crystal Violet staining demonstrates that plaque formation was clearly observed in all five VTT-infected cell lines, but infection with rVTT-TA35-TJ only formed detectable plaques in BHK-21, Hela, and Vero cells. Furthermore, the number of plaques was significantly smaller than in VTT-infected cells (Fig. 4F). The virus weakened the cytopathic effects of the other
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two cells, similar to the results of MTS, indicating that the deletion of the two fragments
significantly reduces the virulence of VTT. These data suggest that rVTT-TA35-TJ is a safer
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vector for vaccination than the wild-type VTT. 3.3 Weight changes in mice and lesion formation in rabbits post infection
Rabbits were inoculated intradermally with rVTT-TA35-TJ and wild type VTT. Each
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concentration was injected into two rabbits and repeated three times. The size of the pock was
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measured by Electronic vernier caliper daily for 18 d. As shown in Figure 5, the pock sizes of
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rVTT-TA35-TJ in every group and low-dose VTT group reached a maximum on the first day post infection, while middle- and high-dose VTT groups reached maximum pock size on the
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fifth and third day post infection. The pock lesions caused by VTT started to fester from the
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third day and gradually formed scabs. By the tenth day, an obvious scab could be observed. At the end of the experiment, the size of the pock lesions induced by VTT were reduced, but they had not all disappeared. In contrast, the pock lesions induced by middle- and high-dose
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rVTT-TA35-TJ groups started to fester from the fourth day, and gradually formed a scab, with the skin lesions disappearing on the ninth day. The size of pock lesions formed by
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rVTT-TA35-TJ infection at the lowest viral titer were significantly smaller than that of lesions formed by VTT infection at the medium and highest titer (P < 0.05). These results demonstrate that the absence of the deleted gene segments led to a reduction in the virulence
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of rVTT-TA35-TJ in small animals. The rVTT-TA35-TJ and wild type VTT viruses were inoculated intranasally into
6-week-old male BALB/c mice, and body weights were recorded every day for 25 days. As shown in Figure 6A, the weight of mice infected with the highest dose of VTT decreased quickly from the fourth day to the ninth day post inoculation (an average reduction of 23%) and started to gradually increase on the tenth day. Until the end of the experiment, the mean
body weight of the VTT group was lower than other groups. The middle-dose VTT group displayed similar results to the high-dose VTT group. However, the low-dose VTT group demonstrates a slowly rising trend. The three rVTT-TA35-TJ groups displayed a similar trend to the PBS control group. In the three rVTT-TA35-TJ groups, the high-dose group incurred more weight loss than the other two groups (P < 0.05). The rVTT-TA35-TJ and wild type VTT viruses were intracranially inoculated into
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3-week-old male BALB/c mice, and the deaths were observed for 14 days post inoculation. From the first day, the VTT groups showed strong neurological symptoms such as lassitude,
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scruffy fur, convulsions, and stiffness. However, the rVTT-TA35-TJ groups did not exhibit such symptoms. Furthermore, mice inoculated with the three different doses of rVTT-TA35-TJ displayed 100% survival (Fig. 6B), which was significantly higher than the
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survival rate of the VTT-infected mice (P < 0.05). The survival rate of the high dose VTT
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group was 0% on the fourth day post inoculation, and the survival rate of low- and
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middle-dose VTT group was 0% at the sixth and eighth day post inoculation. The ICLD50 of VTT-inoculated mice was 1.16 × 104 PFU. These results suggest that the absence of the
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deleted gene segments in rVTT-TA35-TJ significantly reduces the neurotoxicity of the virus
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in mice. The safety of the recombinant vaccinia virus in mice was thus improved by knocking out two genes, which increases its potential as a vaccine vector. 3.4 Cellular and humoral immunity induced by rVTT-TA35-TJ
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The levels of IL-2, IL-4, IL-10, and IFN-γ were measured in all the mice groups with mouse IFN-γ ELISA kit on day 14 and 35 post immunization. These results indicate that the
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levels of IL-2, IL-4, IL-10, and IFN-γ in serum of the rVTT-TA35-TJ and VTT immunized groups were significantly higher than that in the normal saline control group post immunization (Fig. 7A-D, P < 0.01). In contrast, no significant differences were observed
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between the immunization groups (P > 0.05). These results suggest that rVTT-TA35-TJ can induce high levels of IL-2, IL-4, IL-10, and IFN-γ, and maintain its immunogenicity as a vaccine. The mice were inoculated with rVTT-TA35-TJ and VTT at a dose of 106 PFU to detect VTT-specific antibody and neutralizing antibody in the serum. These results are summarized in Figure 7E and 7F. The level of VTT-specific antibody in these immunization groups was
raised on the 14th day post immunization, but it did not significantly increase from day 14 to 28. However, VTT-specific antibody levels also increased at day 35 post immunization. The level of VTT-specific antibody in the immunization groups was significantly higher than in the normal saline control group post immunization (P < 0.01). In contrast, no significant differences were observed between the immunization groups (P > 0.05). The trend of neutralizing antibody titers of the rVTT-TA35-TJ and VTT groups were identical to the trend
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of VTT-specific antibody. Therefore, rVTT-TA35-TJ can stimulate a strong humoral immune
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response.
4. Discussion
The vaccinia virus is relatively safe compared to the human poxvirus, but it inevitably
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causes a series of adverse reactions following the vaccination (Cono et al., 2003; Jacobs et
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al., 2009; Lane and Goldstein, 2003). Although the probability of serious adverse reactions is
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small, its safety still needs to be improved. When vaccinia virus is used as a vaccine vector, severe necrotizing vaccinia can be induced in immunodeficient patients. Therefore, it is
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particularly important for treatment of attenuated vaccinia virus. Currently, the most widely
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used method is to perform gene insertion, deletion, or blockage at a specific position in the viral genome by gene manipulation to obtain an attenuated viral vector. Scientists have been studying the attenuation of Tian Tan strain vaccinia virus for more than 20 years. Wang et al.
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(Wang et al., 2012) established a strain of attenuated Tian Tan strain by knocking out the E3L gene and confirmed good genetic stability. Another study shows that the removal of the viral
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M1L-K2L genes (MVTT2-GFP) was less virulent than VTT by 340-fold, based on the ICLD50 value in mice (Zhu et al., 2007). Furthermore, the removal of the viral C2L to F3L genes (MVTTZCI) attenuated the virus by 1000-fold (Yu et al., 2010).
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In this study, the replication-non-essential gene TJ2R and TA35R of the vaccinia virus
Tian Tan strain was knocked out. The EGFP fluorescent fragment was designed as an exogenous selection marker and TK(TJ2R) was designed as an endogenous selection marker. The Cre/loxP system was introduced into the shuttle plasmid, and the exogenous selection marker was then knocked out. Following continuous screenings and identification, we successfully constructed the TJ2R and TA35R gene deletion VTT attenuated strain:
rVTT-TA35-TJ. The vaccinia virus A35R gene is located in the viral genome at 144 462 to 144 992 bp, is both a virulence and an immunosuppression gene and encodes a 20 kDa protein. Studies have shown that deletion of the A35R gene from the vaccinia virus WR strain genome reduces the virulence approximately 1000 folds compared to the wild virus (Roper, 2006). A35R can prevent antigen presentation and inhibit MHC class II molecules presentation of antigens to
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CD4+ T cells. Following the knock out of the A35R gene, we can increase the immunogenicity of vaccinia virus without affecting the replication ability. Immunization of
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mice with the attenuated virus strain can play a protective role in the wild-virus attack (Rehm and Roper, 2011). Therefore, knocking out the A35R gene reduces the virulence of the
vaccinia virus and increases the immunogenicity, which allows to screen out more attenuated
this
study,
MTS
experiments
demonstrate
that
the
survival
rate
of
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In
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vaccine vectors.
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rVTT-TA35-TJ-infected cells in BHK-21, PK-15, and Vero cells was significantly higher than VTT-infected cells starting at 48 h post infection. Furthermore, and as the infection
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progresses, the differences in survival rate become greater. In Hela and MDCK cells, the
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survival rate of rVTT-TA35-TJ-infected was significantly higher than that of VTT-infected at 96 h post infection. Crystal violet staining reveals identical results. In the BHK-21, Hela, and Vero cells, the rVTT-TA35-TJ infected cells formed detectable plaques, while the other two
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types of cells did not. However, the number of plaques formed was significantly less than in VTT-infected cells. These results indicate that the deletion of the two gene fragments
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significantly reduces the virulence of the vaccinia virus Tian Tan strain, thereby increasing its safety as a vaccine vector. In the in vivo experiments, it was found that the high-dose VTT group reached the lowest
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weight at day 9 post infection, with an average reduction of 23%. The body weight change in the middle-dose VTT group was similar to the high dose group. In contrast, the body weights of the mice in the high, middle, and low dose groups of rVTT-TA35-TJ were not significantly different than the PBS group, indicating that the virulence of the vaccinia virus was greatly reduced after knocking out the two gene fragments. Similarly, it has been reported that inoculation of MVA did not result in a decrease in the weight of the inoculated
mice compared to mice inoculated with the wild-type virus (Melamed et al., 2013). In the skin lesion formation experiment in rabbits, the pocks formed by high-dose VTT infection peaked on day 3 post inoculation, followed by a slow downtrend. In contrast, the rVTT-TA35-TJ group incurred only mild swelling, and the low-dose group recovered on day 13, consistent with the results of previous assays. In addition, the vaccinia virus NYVAC strain was obtained by continuously knocking out
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18 non-essential genes, such as virulence and host-wide genes of the Copenhagen strain. Compared to the wild strain, it has the advantages of low neurotoxicity in suckling mice and
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weak pathogenicity in nude mice (Tartaglia et al., 1992). rVTT-TA35-TJ also displays high
safety in terms of neurotoxicity. After the intracranial injection of the viruses the wild-type group all died on day 8 post infection, while the mice in the rVTT-TA35-TJ high-, medium-,
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and low-dose group survived until the end of the experiment, and with only mild neurological
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symptoms during the first 2 days post inoculation.
Cytokines play an important role in the body’s immune response, such as antiviral and
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mediated inflammatory responses. Research has shown that IL-4, IL-10, and IFN-γ play an
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important role in the immune response of mice to VV infection (van Den Broek et al., 2000).
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Most studies on the deletion of immunoregulatory genes in VV have shown that the absence of several VV genes reduces the toxicity of the virus (Smith et al., 2013), but the effects on immunogenicity is variable. For example, deletion of genes, such as N2L and C16L, was
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found to have no effect on immunogenicity (Alcami and Smith, 1995; Fahy et al., 2008; Ferguson et al., 2013). However, deletion of these immunomodulatory genes (E3L,
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B15R/B16R, A41L, B22R, C12L, and C6L) from different strains (mainly WR and MVA) was found to increase the immunogenicity of this virus. Here, it was found that the recombinant vaccinia virus rVTT-TA35-TJ, lacking the TJ and TA35R gene fragments, had
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no significant difference in immunogenicity level compared to the parental VTT strain. In summary, knocking out the TJ and TA35R gene fragments not only simplified the construction process of the recombinant vaccinia virus, also increased safety compared to VTT, while maintaining its immunogenicity as a vaccine. Therefore, rVTT-TA35-TJ has potential as a new virus vector and vaccine for the prevention or treatment of diseases caused by different pathogens.
Conflicts of interest statement The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Author contributions Conceived and designed the experiments: YQL, XL, LLS and NYJ. Performed the experiments: YQL, SC, JBF, YLZ, BB, WJL, XZY, JW and NYJ. Analyzed the data: YQL, XL, LLS and NYJ. Contributed reagents/materials/analysis tools: JBF, YLZ, XZY, XL and JCH. Wrote the paper: YQL and NYJ. All authors read and approved the final manuscript.
Acknowledgments
This work was supported by the National Science and Technology Major Project (Major New
Key
Research
and
Development
Program
of
China
[grant
number
N
National
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Drugs Innovation and Development) [grant number 2018ZX09301053-004-001], the
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2016YFC1200900] and the Major Technological Program of Changchun City [grant number
M
16ss11].
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REFERENCES
Adelfinger, M., Gentschev, I., Grimm de Guibert, J., Weibel, S., Langbein-Laugwitz, J., Hartl, B., Murua Escobar, H., Nolte, I., Chen, N.G., Aguilar, R.J., Yu, Y.A., Zhang, Q.,
PT
Frentzen, A., Szalay, A.A., 2014. Evaluation of a new recombinant oncolytic vaccinia virus strain GLV-5b451 for feline mammary carcinoma therapy. PloS one 9(8),
CC E
e104337.
Alcami, A., Smith, G.L., 1995. Cytokine receptors encoded by poxviruses: a lesson in cytokine biology. Immunology today 16(10), 474-478.
A
Bray, M., Wright, M.E., 2003. Progressive vaccinia. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 36(6), 766-774.
Casey, C.G., Iskander, J.K., Roper, M.H., Mast, E.E., Wen, X.J., Torok, T.J., Chapman, L.E., Swerdlow, D.L., Morgan, J., Heffelfinger, J.D., Vitek, C., Reef, S.E., Hasbrouck, L.M., Damon, I., Neff, L., Vellozzi, C., McCauley, M., Strikas, R.A., Mootrey, G., 2005. Adverse events associated with smallpox vaccination in the United States,
January-October 2003. Jama 294(21), 2734-2743. Cono, J., Casey, C.G., Bell, D.M., Centers for Disease, C., Prevention, 2003. Smallpox vaccination
and
Recommendations
adverse
reactions.
and reports :
Guidance
for
clinicians.
MMWR.
Morbidity and mortality weekly report.
Recommendations and reports 52(RR-4), 1-28. Eckart, R.E., Love, S.S., Atwood, J.E., Arness, M.K., Cassimatis, D.C., Campbell, C.L.,
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Boyd, S.Y., Murphy, J.G., Swerdlow, D.L., Collins, L.C., Riddle, J.R., Tornberg, D.N.,
Grabenstein, J.D., Engler, R.J., Department of Defense Smallpox Vaccination Clinical
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Evaluation, T., 2004. Incidence and follow-up of inflammatory cardiac complications after smallpox vaccination. Journal of the American College of Cardiology 44(1), 201-205.
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Fahy, A.S., Clark, R.H., Glyde, E.F., Smith, G.L., 2008. Vaccinia virus protein C16 acts
A
general virology 89(Pt 10), 2377-2387.
N
intracellularly to modulate the host response and promote virulence. The Journal of
Ferguson, B.J., Benfield, C.T., Ren, H., Lee, V.H., Frazer, G.L., Strnadova, P., Sumner, R.P.,
M
Smith, G.L., 2013. Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes
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virulence. The Journal of general virology 94(Pt 9), 2070-2081. Garcia-Arriaza, J., Arnaez, P., Gomez, C.E., Sorzano, C.O., Esteban, M., 2013. Improving Adaptive and Memory Immune Responses of an HIV/AIDS Vaccine Candidate
PT
MVA-B by Deletion of Vaccinia Virus Genes (C6L and K7R) Blocking Interferon Signaling Pathways. PloS one 8(6), e66894.
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Jacobs, B.L., Langland, J.O., Kibler, K.V., Denzler, K.L., White, S.D., Holechek, S.A., Wong, S., Huynh, T., Baskin, C.R., 2009. Vaccinia virus vaccines: past, present and future. Antiviral research 84(1), 1-13.
A
Kan, S., Wang, Y., Sun, L., Jia, P., Qi, Y., Su, J., Liu, L., Yang, G., Liu, L., Wang, Z., Wang, J., Liu, G., Jin, N., Li, X., Ding, Z., 2012. Attenuation of vaccinia Tian Tan strain by removal of viral TC7L-TK2L and TA35R genes. PloS one 7(2), e31979. Kemper, A.R., Davis, M.M., Freed, G.L., 2002. Expected adverse events in a mass smallpox vaccination campaign. Effective clinical practice : ECP 5(2), 84-90. Lane, J.M., Goldstein, J., 2003. Adverse events occurring after smallpox vaccination.
Seminars in pediatric infectious diseases 14(3), 189-195. Lederman, E., Miramontes, R., Openshaw, J., Olson, V.A., Karem, K.L., Marcinak, J., Panares, R., Staggs, W., Allen, D., Weber, S.G., Vora, S., Gerber, S.I., Hughes, C.M., Regnery, R., Collins, L., Diaz, P.S., Reynolds, M.G., Damon, I., 2009. Eczema vaccinatum resulting from the transmission of vaccinia virus from a smallpox vaccinee: an investigation of potential fomites in the home environment. Vaccine
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27(3), 375-377.
Li, X., Jin, N., Mi, Z., Lian, H., Sun, L., Li, X., Zheng, H., 2006. Antitumor effects of a
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recombinant fowlpox virus expressing Apoptin in vivo and in vitro. International journal of cancer 119(12), 2948-2957.
Liu, J., McFadden, G., 2015. SAMD9 is an innate antiviral host factor with stress response
U
properties that can be antagonized by poxviruses. Journal of virology 89(3),
N
1925-1931.
A
Mackett, M., Yilma, T., Rose, J.K., Moss, B., 1985. Vaccinia virus recombinants: expression of VSV genes and protective immunization of mice and cattle. Science 227(4685),
M
433-435.
ED
MacNeil, A., Reynolds, M.G., Damon, I.K., 2009. Risks associated with vaccinia virus in the laboratory. Virology 385(1), 1-4.
Melamed, S., Wyatt, L.S., Kastenmayer, R.J., Moss, B., 2013. Attenuation and of
PT
immunogenicity
host-range
extended
modified
vaccinia
virus
Ankara
recombinants. Vaccine 31(41), 4569-4577.
CC E
Meulemans, G., Letellier, C., Gonze, M., Carlier, M.C., Burny, A., 1988. Newcastle disease virus F glycoprotein expressed from a recombinant vaccinia virus vector protects
A
chickens against live-virus challenge. Avian pathology : journal of the W.V.P.A 17(4), 821-827.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immunological methods 65(1-2), 55-63. Noisumdaeng, P., Pooruk, P., Kongchanagul, A., Assanasen, S., Kitphati, R., Auewarakul, P., Puthavathana, P., 2013. Biological properties of H5 hemagglutinin expressed by
vaccinia virus vector and its immunological reactivity with human sera. Viral immunology 26(1), 49-59. Pastoret, P.P., Brochier, B., 1996. The development and use of a vaccinia-rabies recombinant oral vaccine for the control of wildlife rabies; a link between Jenner and Pasteur. Epidemiology and infection 116(3), 235-240. Qin, L., Favis, N., Famulski, J., Evans, D.H., 2015. Evolution of and evolutionary
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relationships between extant vaccinia virus strains. Journal of virology 89(3), 1809-1824.
SC R
Reed LJ, M., H, 1938. A simple method of estimating fifty percent endpoints. Am J Hyg 27, 493–497.
Rehm, K.E., Roper, R.L., 2011. Deletion of the A35 gene from Modified Vaccinia Virus
U
Ankara increases immunogenicity and isotype switching. Vaccine 29(17), 3276-3283.
N
Remy-Ziller, C., Germain, C., Spindler, A., Hoffmann, C., Silvestre, N., Rooke, R.,
A
Bonnefoy, J.Y., Preville, X., 2014. Immunological characterization of a modified vaccinia virus Ankara vector expressing the human papillomavirus 16 E1 protein.
M
Clinical and vaccine immunology : CVI 21(2), 147-155.
ED
Rintoul, J.L., Wang, J., Gammon, D.B., van Buuren, N.J., Garson, K., Jardine, K., Barry, M., Evans, D.H., Bell, J.C., 2011. A selectable and excisable marker system for the rapid creation of recombinant poxviruses. PloS one 6(9), e24643.
PT
Roper, R.L., 2006. Characterization of the vaccinia virus A35R protein and its role in virulence. Journal of virology 80(1), 306-313.
CC E
Smith, G.L., Benfield, C.T., Maluquer de Motes, C., Mazzon, M., Ember, S.W., Ferguson, B.J., Sumner, R.P., 2013. Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity. The Journal of general virology 94(Pt 11), 2367-2392.
A
Tartaglia, J., Perkus, M.E., Taylor, J., Norton, E.K., Audonnet, J.C., Cox, W.I., Davis, S.W., van der Hoeven, J., Meignier, B., Riviere, M., et al., 1992. NYVAC: a highly attenuated strain of vaccinia virus. Virology 188(1), 217-232. van Den Broek, M., Bachmann, M.F., Kohler, G., Barner, M., Escher, R., Zinkernagel, R., Kopf, M., 2000. IL-4 and IL-10 antagonize IL-12-mediated protection against acute vaccinia virus infection with a limited role of IFN-gamma and nitric oxide synthetase
2. Journal of immunology 164(1), 371-378. Wang, Y., Kan, S., Du, S., Qi, Y., Wang, J., Liu, L., Ji, H., He, D., Wu, N., Li, C., Chi, B., Li, X., Jin, N., 2012. Characterization of an attenuated TE3L-deficient vaccinia virus Tian Tan strain. Antiviral research 96(3), 324-332. Wollenberg, A., Engler, R., 2004. Smallpox, vaccination and adverse reactions to smallpox vaccine. Current opinion in allergy and clinical immunology 4(4), 271-275.
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Yu, W., Fang, Q., Zhu, W., Wang, H., Tien, P., Zhang, L., Chen, Z., 2010. One time intranasal
vaccination with a modified vaccinia Tiantan strain MVTT(ZCI) protects animals
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against pathogenic viral challenge. Vaccine 28(9), 2088-2096.
Zhu, W., Fang, Q., Zhuang, K., Wang, H., Yu, W., Zhou, J., Liu, L., Tien, P., Zhang, L., Chen, Z., 2007. The attenuation of vaccinia Tian Tan strain by the removal of the viral
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M1L-K2L genes. Journal of virological methods 144(1-2), 17-26.
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Figure Legends:
Fig. 1. Construction of an attenuated vaccinia virus, rVTT-TA35-TJ. Structure of the plasmid vector (A), recombinant vaccinia virus (B), and gene-deleted virus
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(C). EGFP was inserted as a screening marker at the TJ2R gene site of VTT (A) and driven by the synthetic early/late promoter (pE/L) to obtain the recombinant virus with deletion of
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the TJ2R gene, but containing the EGFP gene (B). The recombinant virus rVTT-J was generated through Cre/loxP site-specific recombination to remove the EGFP gene (C). The same
method
was
used
to
successively
knock
out
TA35R.
Ultimately,
the
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two-segment-deleted attenuated strain rVTT-TA35-TJ was obtained. T, termination signal of vaccinia virus; loxP, loxP site; Cre, Cre recombinase.
Fig. 2. Screening and identification of mutants. The fused EGFP was expressed by the mutants rVTT-J-EGFP (A1) and rVTT-A35-J-EGFP (B1) in BHK-21 cells. The virus-infected cells were identified by visualization of isolated
fluorescent plaques in the same visual fields. Non-fluorescent plaques were observed for rVTT-J(A2) and rVTT-TA35-TJ (B2) mutants in BHK-21 cells (magnification 200×, A–B); PCR was performed to identify the final mutant rVTT-TA35-TJ (C-F). (C) Lane 2: positive control containing the partial segments TA35R gene (307 bp), lane 4: positive control containing the partial segments TJ2R gene (261 bp), and lanes 1 and 3: PCR products of the rVTT-TA35-TJ genome; (D) Lane 1: positive control containing the TA35R gene (751 bp),
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Lane 2: PCR products of the rVTT-TA35-TJ genome; (E) Lane 1: positive control containing the TJ2R gene (551 bp), Lane 2: PCR products of the rVTT-TA35-TJ genome; Evaluation of
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the genetic stability of the recombinant virus by PCR. (D) TA35R gene of rVTT-TA35-TJ (lanes 2–5) and (E) TJ2R gene of rVTT-TA35-TJ (lanes 2–5). PCR of all gene-deleted mutants produced negative results, compared to the corresponding positive PCR controls
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(lane 1).
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Fig. 3. In vitro replication of rVTT-TA35-TJ and VTT.
Growth curve of rVTT-TA35-TJ and VTT in BHK-21 (A), MDCK (B), Hela (C), Vero (D),
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and PK-15 (E) cells. Cells were infected with rVTT-TA35-TJ and VTT at an MOI of 0.5, and
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the virus titers were measured at 3, 12, 24, and 48 h post infection.
Fig. 4. In vitro cytotoxicity of rVTT-TA35-TJ and VTT.
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BHK-21 (A), MDCK (B), Hela (C), Vero (D), and PK-15 (E) cells were seeded in 96-well plates (1 × 104 cells/well) and infected with rVTT-TA35-TJ and VTT at an MOI of 0.5. Cell
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viability was measured every 24 h over a 96 h period. All measurements were performed in triplicate. Data are presented as the mean ± standard deviation. Confluent monolayers of BHK-21, MDCK, Hela, Vero, and PK-15 cells in 12-well plates were infected with 0.5 MOI
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of rVTT-TA35-TJ and VTT, then stained with 0.4% crystal violet after plaque phenotypes formed. The pathogenicity of rVTT-TA35-TJ decreased in all five cell lines (compared with VTT as the control) (F).
Fig. 5. Virulence of rVTT-TA35-TJ or VTT in rabbits. Rabbits were infected with 105, 106, or 107 PFU of rVTT-TA35-TJ or VTT (as a positive
control) or PBS (as a negative control) by intradermal injection. Infection with 10 5, 106, and 107 PFU of rVTT-TA35-TJ resulted in mild swelling by day 2, and no visible scars were observed by day 10 at the sites of infection (A). However, infection with VTT resulted in the formation of severe lesions at the infection site, and the appearance of ulcerations. The size of the lesions increased until they reached a maximum at day 3, after which they gradually decreased, but left scars (A). The diameter of the lesions was associated with the VTT and
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rVTT-TA35-TJ dose (B).
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Fig. 6. Virulence of VTT and rVTT-TA35-TJ in mice after intranasal and intracranial infection.
(A) Body weights were monitored in mice that were intranasally infected with 105, 106, or
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107 PFU of rVTT-TA35-TJ or VTT (as a positive control) or PBS (as a negative control).
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Error bars indicate the standard error of the mean, and differences between groups were
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determined by two-way repeated measures analysis of variance. Mice inoculated with VTT showed significant signs of illness, and a clear positive correlation was found between the
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viral dose and weight loss (P < 0.05). No distinct weight changes or signs of illness were observed in the animals inoculated with rVTT-TA35-TJ or PBS.
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(B) Mice were inoculated intracranially with 105, 106, or 107 PFU of rVTT-TA35-TJ or VTT (PBS in the negative control group), and the survival rates were observed for 14 days. All the
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mice inoculated with rVTT-TA35-TJ survived, while all the mice infected with VTT died.
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Fig. 7. Immune responses to rVTT-TA35-TJ or VTT in vaccinated mice. BALB/c mice were inoculated intramuscularly with 106 PFU rVTT-TA35-TJ or VTT, and given a booster immunization of the same dose in 0.1 ml PBS three weeks later. The serum
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samples were harvested at weeks 3 and 5 post immunization. The IL-2 (A), IL-4 (B), IL-10 (C), and IFN-γ (D) levels in the two vaccinated groups were measured by mouse ELISA kits. All measurements were performed in triplicate. Data are presented as the mean ± SD values. VTT-specific IgG levels were measured by ELISA on VTT-coated plates and then read by a microplate reader. Data are presented as mean ± SD (E). The neutralization antibody titer was calculated by determining the highest serum dilution required to generate a 50% viral plaque
reduction (F). All measurements were performed in triplicate. Data are presented as the mean
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