key: cord-0689897-sgpktv4g
authors: Zhu, Weijun; Fang, Qing; Zhuang, Ke; Wang, Haibo; Yu, Wenbo; Zhou, Jingying; Liu, Li; Tien, Po; Zhang, Linqi; Chen, Zhiwei
title: The attenuation of vaccinia Tian Tan strain by the removal of the viral M1L-K2L genes
date: 2007-04-24
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2007.03.012
sha: b715b120180b24a6efb23ca38fb3bdea33ac3e40
doc_id: 689897
cord_uid: sgpktv4g

To generate a safe vaccinia Tian Tan (VTT)-based vaccine vector, it is necessary to develop a method to attenuate the virus. A modified VTT (MVTT(2-GFP)) was constructed by replacing the viral M1L-K2L genes with a GFP gene. In comparison to the parental VTT, MVTT(2-GFP) lost its replication capacity in rabbit RK13 and human HeLa cell lines. The life cycle of viral replication was blocked at different stages in these two cell lines as determined by electron microscope examination. MVTT(2-GFP) was less virulent than VTT for 100-fold by measuring mouse body weight loss after intranasal viral inoculation and for 340-fold by determining the intracranial LD(50) value in mice. The foreign GFP gene was stable genetically after 10 rounds of passage in Vero cells. Importantly, MVTT(2-GFP) elicited both humoral and cell-mediated immune responses to the GFP gene in mice. With two intramuscular inoculations of 10(5) PFU virus, the anti-GFP antibody reciprocal endpoint titer reached over 700 as determined by an ELISA. The number of IFN-γ secreting T cells reached over 350 SFU per million splenocytes against a CD8+ T cell-specific epitope of GFP. Collectively, the removal of the M1L-K2L genes is a useful method to generate an attenuated vaccinia Tian Tan vaccine vector.

After the declaration of the worldwide eradication of smallpox in 1980, vaccinia virus has been studied for use as a live viral vector for other infectious diseases or cancer therapy (Moss, 1996; Mwau et al., 2004; Paoletti, 1996) . In the People's Repub-Abbreviations: VTT, vaccinia Tian Tan; MVTT, modified vaccinia Tian Tan; MVA, modified vaccinia Ankara; GFP, green fluorescent protein; AIDS, acquired immunodeficiency syndrome; HIV-1, human immunodeficiency virus type one; SIV, simian immunodeficiency virus; SHIV, simian/human immunodeficiency chimeric virus; CTL, cytotoxic T lymphocytes; MOI, multiplicity of infection; PFU, plaque forming unit; p.i., post-infection; EM, electron microscope lic of China, the vaccinia Tian Tan (VTT) strain was used historically as a smallpox vaccine for millions of people, yet its potential as a vaccine vector has not been explored carefully. The full-length sequence of the VTT genome was determined by Jin et al. (1997) . Based on sequence comparison, VTT displays genetic features distinct from other vaccinia viruses including the vaccinia western reserve (WR) strain (Hou et al., 1985; Jin et al., 1997; Tsao et al., 1986) . In particular, unique deletions or insertions, which affect multiple genes, were identified in the restriction enzyme HindIII-C, B and A fragments. These data infer the plausibly distinct biological properties of VTT (Jin et al., 1997) . Recently, the biological properties of VTT were characterized in terms of its host cell range and growth properties in vitro and virulence in vivo (Fang et al., 2005) . Although VTT is significantly less virulent than the vaccinia WR strain, it remains lethal in mice after intracranial inoculation and causes significant body weight loss after intranasal inoculation (Fang et al., 2005) . These properties have limited its use as a vaccine vector for human use, especially for immune-suppressed individuals. Therefore, further attenuation of VTT is necessary for the development of a useful vaccine vector.

Various viral vector systems have been evaluated for vaccine development Santra et al., 2005; Tartaglia et al., 1992) . The poxvirus vector is the live recombinant vector which has been studied most intensively. The widely studied vaccinia-based vector is probably the modified vaccinia virus Ankara (MVA). This vector is safe in humans, even in some immunocompromised individuals (Cosma et al., 2003) . Moreover, MVA-based vaccines are effective for inducing protective responses against different viruses, such as severe acute respiratory syndrome coronavirus, influenza and respiratory syncytial virus (Bisht et al., 2004; Chen et al., 2005; De Waal et al., 2004; Degano et al., 1999; Olszewska et al., 2004; Sutter et al., 1994; Wyatt et al., 1999) . MVA-based vaccines are also effective in delaying the development of AIDS and the progression of the disease in rhesus monkeys infected with simian immunodeficiency virus (SIV) or simian/human immunodeficiency virus (SHIV) (Barouch et al., 2001) . However, the immunogenicity profile of MVA as a vaccine vector for HIV-1 is unsatisfactory in humans. A recent phase one trial indicated that an MVA-based HIV-1 vaccine is not very immunogenic in humans (Goonetilleke et al., 2006) . It is, therefore, desirable to explore the potential of other vaccinia-based vector systems for stimulating stronger host immune responses in humans.

In this study, a method to generate a modified VTT (MVTT 2-GFP ) is described. This method involves the genetic modification of the parental VTT genome by deleting the M1L-K2L genes including one host range gene, K1L. The rationale for choosing these genes for deletion corresponds to the lost genes for the deletion II region of MVA genome (Antoine et al., 1998; Meyer et al., 1991) . Before this study, it was unknown to what extent a vaccinia virus could be attenuated just by removing the M1L-K2L genes. As the deletion II region of MVA serves as an ideal insertion site for foreign genes, the effectiveness of the corresponding region in the VTT genome for the expression of a foreign gene was also unknown. For these reasons, the phenotypic changes of MVTT 2-GFP in comparison to the parental VTT both in vitro and in vivo, and the potential use of MVTT 2-GFP as a live viral vector for vaccine development were examined in this study.

Thirteen cell lines of various origins were maintained under growth conditions suggested by the American Type Culture Collection (ATCC, Rockville, MD). These cell lines include COS-7, WISH, BHK-21, MRC-5, CHO-K1, HeLa, 293T, RK(15), MDCK, RK13, C6, Vero and NIH/3T3. Primary chicken embryo fibroblasts (CEF) were prepared from 10-day-old embryos. The parental vaccinia Tian Tan 761 strain was obtained from the Institute of Virology at the Chinese Center for Disease Control and Prevention. This VTT 761 strain was derived originally from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (NICPBP). Virus stocks were propagated in Vero cells and then purified by centrifugation through a 36% sucrose cushion. The virus stocks were titrated by a plaque forming assay using crystal violet staining.

The shuttle vector was constructed by inserting two genome fragments flanking the deletion region of VTT. The GFP gene was placed under a synthetic early/late promoter P syn (Chakrabarti et al., 1997; Wyatt et al., 1996) . The MVTT 2-GFP virus was made by a homologous recombination method as described previously (Sutter and Moss, 1992; Sutter et al., 1994; Wyatt et al., 1996) . The plaques that express GFP were selected and purified subsequently through six rounds of plaque purification under agarose. The insert in the recombinant virus was determined by a nested PCR using primer pairs flanking the deletion region. PCR primers used for this test were: 5 -GTTTATACAATCCATGCTACTACCTTCGGG-3 (sense) and 5 -GAATTTCCATTACATCAGGCAGCCACATTGGAAG-3 (antisense) for the first round reaction and 5 -GGTTCATTG-TTATCCATTGCAGAGGACG-3 (sense) and 5 -GTAAAGA-TTTTGCTATTCAGTGGACTGGATG-3 (antisense) for the second round of reaction. The amplification cycles are 95 • C for 2 min followed by 35 cycles of 94 • C for 20 s, 52 • C for 45 s and 72 • C for 3 min plus the last extension of 72 • C for 8 min. Amplified PCR products were purified using a QIAquick PCR purification kit (QIAgen, Valencia, CA, USA) and were subjected to DNA sequencing directly by an automated ABI 377 DNA sequencer (Applied Biosystems Inc., Foster City, CA, USA). For comparison, a recombinant VTT GFP was generated by placing GFP gene in a location upstream of the hemagglutinin gene without artificial interruption of the M1L-K2L genes.

Assays for the determination of viral host cell range, growth property in vitro and virulence of MVTT 2-GFP in vivo have been described previously (Carroll and Moss, 1997; Fang et al., 2005) .

Confluent cell monolayers were infected with 5 MOI of MVTT2-GFP or VTT. The virus was allowed to attach to the cells for 90 min at 37 • C. The cells were then washed with medium three times and incubated at 37 • C for an additional 16 h. The infected cells were then trypsinized to detach them from the culture plates and washed with PBS twice. The cell pellets were fixed in 2.5% glutaraldehyde and processed for examination using a transmission electron microscopy by a routine technique described previously (Wolffe et al., 1996) .

After a six-round purification of the GFP positive plaques, ten additional rounds of passage of the virus were carried out. After the last passage, Vero cells were infected subsequently with MVTT 2-GFP at an MOI of 0.01. Forty-eight hours after infection, the percentages of GFP and vaccinia antigen double positive plaques were counted. The method for the immunostaining of vaccinia specific antigens has been described previously (Carroll and Moss, 1997) .

BALB/c mice were immunized with MVTT 2-GFP . Groups of three 6-8-week-old female BALB/c mice were immunized intramuscularly twice with 10 5 PFU of MVTT 2-GFP or the parental type VTT in 100 l of PBS at weeks 0 and 3. Serum samples were collected at weeks 0, 3 and 5 for anti-GFP antibody testing (Gambotto et al., 2000; Hsu et al., 2005) . The anti-GFP antibody endpoint titer was determined as the reciprocal highest dilution of samples producing at least two-fold greater than the value corresponding to the optical density readout of the control serum sample at the same dilution. The coating antigen was recombinant His-tagged EGFP that was purified from E. coli by nickel column chromatography. At week 5, the mice were killed and splenocytes were isolated from the mice for measuring the cellmediated immune response by an ELISpot assay Kit (U-Cytech, The Netherlands). A CD8+-specific epitope HYLSTQSAL was used as the pulsing antigen (Gambotto et al., 2000) . The mice received PBS served as the negative controls. Staphylococcus aureus enterotoxin B (SEB) was used as a positive control. Spots were counted by using an ELISpot reader (BIOREADER 4000, Bio-sys Inc., Germany).

Serial two-fold dilutions of heat-inactivated mouse serum were incubated with 100 PFU of the parental VTT at 37 • C for 1 h. The mixed solution was then inoculated onto Vero cells in 24-well plates. The cells were over-layered by agarose after 90 min of viral absorption. After 48 h of incubation at 37 • C, the viral plaques were visualized by crystal violet staining and counted. Antibody titers were defined as the reciprocal of serum dilution that reduced viral plaques by 50%. Serum samples from pre-immunized or mock-immunized animals were included as background controls.

Using a homologous recombination method, a GFP gene was introduced into the VTT genome to generate the MVTT 2-GFP . The introduction of the GFP gene replaced a fragment of viral genome from position 22,372 to 25,187 bases of VTT (Gen-Bank accession number AF095689). This replacement resulted in the deletion of viral M1L-K2L genes (Tan-TM1L, Tan-TM2L, Tan-TK1L and Tan-TK2L) ( Table 1 ) (Drillien et al., 1981) . This replacement was successful as the plaque-purified GFP positive virus was tested by a specific PCR, immunochemical staining and subsequent sequence analysis (data not shown). After a series of plaque purifications, a pure viral stock of MVTT 2-GFP was generated as determined by the co-expression of GFP and vaccinia specific antigens (data not shown).

To determine the effect of these deleted VTT genes on the viral biological properties, the growth kinetics of MVTT 2-GFP were quantitatively characterized using a previously described method (Carroll and Moss, 1997) . Thirteen mammalian cell lines of various host origin and primary chicken embryo fibroblasts (CEF) were infected with MVTT 2-GFP at an MOI of 0.05. These cell lines included COS-7, WISH, BHK-21, MRC-5, CHO-K1, HeLa, 293T, RK(15), MDCK, RK13, C6, Vero and NIH/3T3. Since these cells were derived from diverse host and tissue origins, this experiment determined the breadth of the host cell range of MVTT 2-GFP . In comparison to the parental VTT, the replication property of MVTT 2-GFP did not change significantly in cell lines including COS-7, Vero, WISH, BHK-21, MRC-5, CHO-K1, NIH/3T3 and CEF. For example, MVTT 2-GFP replicates well in Vero cells (Fig. 1 ). However, the replication level of MVTT 2-GFP dropped more than 6-10-fold in cell lines including MDCK, C6, RK13, PK(15), 293T and HeLa ( Fig. 1 and Table 2 ). In particular, MVTT 2-GFP did not seem to replicate in RK13 and HeLa cells overtime. These results indicate that the loss of viral M1L-K2L genes likely has a significant impact on the growth capacity of the virus in certain cell lines in vitro. VV-Tan-TK1 L Early Ankyrin/host range Chung et al. (1997) , Gillard et al. (1986) , Perkus et al. (1990) , Ramsey-Ewing and Moss (1996) and Shisler and Jin (2004) K2L VV-Tan-TK2L Intermediate Serine proteases inhibitor (SPI), prevent cell fusion Gambotto et al. (2000) , Hsu et al. (2005) , Law and Smith (1992) , Turner and Moyer (1995) and Zhou et al. (1992) (Carroll and Moss, 1997) . Cells were infected with an MOI of 0.01. b Virus replication (fold increase in virus titer) determined by dividing the virus yield at 72 h by the input titer. Cell lines were therefore categorized into permissive (P, >25-fold increase), semi-permissive (SP, 1-25-fold increase) and non-permissive (NP, <1-fold increase) (Carroll and Moss, 1997) . Cells were infected with an MOI of 0.05.

An immunostaining analysis on cells infected with MVTT 2-GFP was conducted to determine the degree of cellto-cell spread in order to further understand the underlined mechanism of reduced viral growth capacity. All 13 mammalian cell lines and CEF became infected with MVTT 2-GFP . The infection was indicated by the expression of specific antigens stained with a polyclonal anti-vaccinia serum in infected cells (Fig. 2) . When compared with the parental VTT strain, the MVTT 2-GFP spread well in cell lines including COS-7, Vero, WISH, BHK-21, MRC-5, NIH/3T3 and CEF. MVTT 2-GFP , however, displayed restricted spread in several cell lines including MDCK, C6 and 293T (Fig. 2) . The fewer number of positive cells found in each foci at 48 h p.i. indicates that the viral spread via cellto-cell contact was less efficient. The reduced spread capacity of MVTT 2-GFP via cell-to-cell contact was likely a reason for the decreased replication kinetics in these cell lines.

To understand further the viral life cycle in RK13 and HeLa cells, MVTT 2-GFP and the parental VTT were subjected to the EM examination. By examining the morphology of newly produced viral particles, the stage of viral replication blockade in these cell lines was expected to be resolved. RK13 and HeLa cells were analyzed around 16 h p.i. (Fig. 3) . As shown in Fig. 3A , neither intracellular mature virions (IMVs) nor immature virions (IVs) were found in RK13 cells infected with MVTT 2-GFP . However, HeLa cells showed different results (Fig. 3B) . Immature virions were often found in the cytoplasm of HeLa cells whereas few IMVs could be identified. As controls, both cell lines produced the majority of the viral particles with characteristic brick-shape of IMV in their cytoplasm after they were infected with the parental VTT ( Fig. 3C and D) . Therefore, the data indicated that the aborted viral replication in these two cell lines was likely due to blockades at different stages of viral replication, before the assembly of immature virions in RK13 cells and around the maturation time of IMV morphogenesis in HeLa cells.

The in vivo virulence of MVTT 2-GFP was evaluated in inbred BALB/c mice using methods as described previously (Lee et al., 1992; Betakova et al., 2000; Zhang et al., 2000) . The viral virulence was first determined by the daily measurement of body weight change for a period of 10 days after the animals were inoculated with the virus via the intranasal route. Five mice were tested for each of the three dose groups: 10 4 , 10 5 and 10 6 PFU per mouse. As shown in Fig. 4 , none of the infected mice died during the experiment. Although mice infected with MVTT 2-GFP showed less than 10% of weight loss over 7 days in the high dose group (10 6 PFU), the virus is significantly less virulent than the parental VTT, which resulted in about 30% weight loss during the same period of time (Fang et al., 2005) . Interestingly, the body weight change in mice infected with 10 6 PFU of MVTT 2-GFP was similar to that of mice that were given 10 4 PFU of parental VTT (Fig. 4) . These results suggest that MVTT 2-GFP was attenuated for about 100-fold in this setting.

Subsequently, the neurovirulence of MVTT 2-GFP was evaluated by infecting BALB/c mice via the intracranial route. The neurovirulence in infected mice was determined by measuring 50% of the intracranial lethal infectious dose (ICLD 50 ). Six 4-week-old mice per group were inoculated with a series of five-fold diluted viruses starting from 3 × 10 6 PFU. By counting the number of dead animals daily (Fig. 5) , the ICLD 50 was calculated using the Reed-Muench method (Reed and Muench, 1938) . ICLD 50 of MVTT 2-GFP was 1.05 × 10 6 PFU. Since the ICLD 50 of the parent VTT is 3.1 × 10 3 PFU, these results demonstrated that MVTT 2-GFP has been attenuated for about 340-fold.

A series of passages of the virus were conducted in Vero cells in order to determine whether MVTT 2-GFP could be developed as a novel vaccine vector. After 10 rounds of passage, anti-vaccinia antibodies were used to stain the infected foci. By evaluating 98 foci, all displayed GFP expression (data not shown). This finding indicates that the foreign GFP gene is stable in the genome of MVTT 2-GFP . This pure GFP-expressing virus may enable the generation of reporter gene-naïve MVTT 2-GFP -based vaccines through reverse GFP selection. This is crucial for eliminating the redundant reporter gene in vaccines for human use.

Groups of BALB/c mice were inoculated with the virus to evaluate how well MVTT 2-GFP could be used as a vaccine vector. The immune responses against the foreign protein GFP were subsequently determined by an ELISA against purified recombinant EGFP. MVTT 2-GFP induced a GFP specific antibody response. The response was detected after the first immunization with an average serum reciprocal titer of 250 (Fig. 6A) . After the second immunization, there was a significant boost effect. The average antibody response to GFP was doubled and the average serum reciprocal titer reached 700. The level of this antibody response was similar to that induced by a control recombinant VTT GFP (Fig. 6A) . Moreover, the cell-mediated immune response to GFP was also detected by an ELISpot assay. Approximately 350 spot-forming cells (SFC) per million splenocytes were detected against a specific CTL epitope HYL-STQSAL (Fig. 6B) . Immunization with VTT GFP also induced a Fig. 7 . Neutralizing antibody responses elicited against VTT in mice immunized with MVTT 2-GFP or VTT. Serum samples collected at weeks 3 and 5 p.i. were tested. Antibody titers were defined as the reciprocal of serum dilution that reduced viral plaques by 50%. Bars represent the average values of three samples (±standard deviations). Serum samples from pre-immunized or mockimmunized animals were included as background controls. This experiment was repeated twice with similar results obtained.

cell-mediated immune response to GFP but at a slightly lower level. In contrast, immunization with wild type VTT or placebo did not induce any immune responses to GFP. These data indicate that MVTT 2-GFP is capable of eliciting specific immune responses against foreign antigens in vivo likely with a comparable potency of VTT GFP .

In addition to the immune responses against GFP, the neutralizing antibody titer against the parental vector VTT was also evaluated. Consistent with the antibody response against GFP, the level of neutralizing antibody response against VTT was boosted significantly after the second immunization (Fig. 7) . A slightly higher level of neutralizing antibodies was induced Fig. 6 . Humoral and cell-mediated immune responses elicited against GFP in mice immunized with MVTT 2-GFP . Mice were intramuscularly inoculated with 10 5 PFU of MVTT 2-GFP or VTT and boosted at a 3-week interval. Mice that received PBS served as controls. (A) Sera were tested for specific IgG antibodies against GFP by ELISA at day 0, weeks 3 and 5 p.i. Responses in naïve (week 0) animals are not shown as the absorbance readouts of those samples fell below the cutoff values that were used for the determination of antibody titer. The differences between MVTT 2-GFP and VTT at test time points are statistically significant (p < 0.01). (B) The number of IFN-␥-secreting CD8+ T cells specific for a GFP epitope HYLSTQSAL was determined by an ELIspot assay. Bars represent the average values of three samples (±standard deviations). The difference between MVTT 2-GFP and VTT at week 5 p.i. is statistically significant (p < 0.01). These experiments were repeated twice with similar results obtained. by VTT than by MVTT 2-GFP . This difference, however, did not reach statistical significance. Therefore, the removal of the M1L-K2L genes did not seem to affect the major neutralizing determinants of vaccinia virus.

MVTT 2-GFP displayed distinct biological properties when compared with the parental VTT. First, MVTT 2-GFP displayed a reduced replication capacity in two human cell lines, HeLa and 293T, as well as in animal cell lines including PK(15), RK13, C6 and MDCK. These phenotypic changes were found to be likely related to the reduced replication and cell-to-cell spread capacity of MVTT 2-GFP . The RK13 and HeLa cell lines became non-permissive to MVTT 2-GFP infection. The EM examination indicated that the life cycle of MVTT 2-GFP was significantly blocked at a relatively early stage before the assembly of immature virions in RK13 cells but at a stage of virion maturation in HeLa cells. Second, MVTT 2-GFP is much less virulent as determined by two separated experiments. About 10% body weight loss was found at day 7 p.i. in mice that received the high dose (10 6 PFU) of MVTT 2-GFP via the intranasal route of inoculation. Since 10 4 PFU of the parental VTT caused a similar level of body weight loss, MVTT 2-GFP was likely attenuated by about 100-fold. Moreover, MVTT 2-GFP displayed an increase of intracranial LD 50 in mice via the intracranial inoculation that was 340 times greater than the parental VTT. These findings indicate that MVTT 2-GFP is an attenuated vaccinia strain. This attenuation is essential for its future development as a safe vaccine vector (Lee et al., 1992; Moss, 1996; Tartaglia et al., 1992) . This attenuation, however, does not seem to affect the expression of the foreign antigens GFP in various cell types infected individually. This property is a prerequisite for a vaccinia-based vaccine vector. The GFP gene in MVTT 2-GFP may serve either as a reverse selection marker for vaccine construction or as a reporter in a neutralization assay.

The phenotypic changes observed in MVTT 2-GFP are related to the loss of the M1L-K2L genes in the VTT genome. Previous studies revealed that VTT displays genetic features distinct from other vaccinia viruses (Hou et al., 1985; Tsao et al., 1986) . These genetic features led to distinct biological characteristics of VTT when compared to vaccinia WR strain (Fang et al., 2005) . In this study, four vaccinia genes including three early genes, M1L, M2L and K1L, as well as one intermediate gene, K2L were removed from the VTT genome to generate MVTT 2-GFP . These four genes have not been found associated with the virulence of a vaccinia virus in vivo before. Of them, K1L is a previously defined host range gene that is essential for vaccinia viral replication in RK13 cells (Chung et al., 1997; Perkus et al., 1990; Ramsey-Ewing and Moss, 1996) . Consistent with previous findings for VVdelK1L and MVA, MVTT 2-GFP replicated poorly also in RK13 cells. Since the K1L gene inhibits the apoptosis of RK13 cells and host NF-kappaB activation (Shisler and Jin, 2004) , a separate study is necessary to address whether or not the K1L gene of VTT alone would have similar biological functions. The K2L gene encodes a serine protease inhibitor which inhibits cell-cell fusion (Law and Smith, 1992; Turner and Moyer, 1995; Zhou et al., 1992) . The deletion of K2L alone did not change virus replication in vitro as well as the virulence and immunogenicity in vivo (Law and Smith, 1992; Turner and Moyer, 1995; Zhou et al., 1992) . The loss of K2L gene in MVTT 2-GFP does not induce cell-cell fusion in every cell type tested (Fig. 3) . Consistent with previous findings, the property of enhanced cell fusion does not increase the virulence of MVTT 2-GFP in vivo because the virus was attenuated instead. To date, the biological functions of vaccinia M1L and M2L genes remain not clear. M1L was suggested previously to function as an ankyrin-like protein (Shchelkunov et al., 1993) . At this stage, although the loss of M1L-K2L genes has resulted in the formation of an attenuated MVTT 2-GFP , whether or not each of the four genes has contributed to the phenotypic changes remains exclusive. Further analysis of each individual gene is required to obtain a definitive answer. Nevertheless, the loss of the M1L-K2L genes does not affect the replication of MVTT 2-GFP in Vero and CEF cells. Since other attenuated viruses (e.g. MVA) rely solely on CEF cells for production, the growth property of MVTT 2-GFP in Vero cells may offer an advantage for future manufacture because Vero is one of the WHO and FDA approved cell lines for vaccine production. As for the development of a human vaccine vector, it is anticipated that the further inactivation of additional VTT genes involved in host cell range (e.g. C7L) and in viral virulence (e.g. hemagglutinin, TK, etc.) would be necessary (Antoine et al., 1996; Shida et al., 1988) .

MVTT 2-GFP elicits specific immune response against the foreign antigen GFP. To use an attenuated vaccinia virus as a live vaccine vector, one critical question is whether or not this virus is still immunogenic for inducing specific immune response against foreign antigens. To address this question, the immune response against the vector and foreign antigen was determined. With a suboptimal dose of 10 5 PFU, MVTT 2-GFP and VTT induced similar levels of neutralizing antibody responses to VTT (Fig. 7) . GFP-specific humoral and cell-mediated immune responses were detected. The levels of these responses are comparable to those induced by a VTT GFP virus. Since GFP is apparently stable in the genome of MVTT 2-GFP , this virus can be developed further to become a live vaccine vector for infectious pathogens or tumors. In this case, the genes encoding the pathogen or tumor specific antigens can be placed in the genomic location of the GFP gene. In future studies, a comparative study using MVTT 2-GFP and other vaccinia strains (e.g. MVA) is essential to reveal the fundamental differences or advantages of these systems to serve as vaccine vectors. Considering that MVTT 2-GFP remains replication-competent in several mammalian cells tested, a correlation between dosing and vaccine efficacy needs to be evaluated, as would the non-invasive mucosal routes of vaccination. The latter is critical because the inoculation of vaccinia-based vaccine via mucosal route can induce immune response against foreign genes even in the presence of preexisting anti-vector immunity (Belyakov et al., 1999) . It was demonstrated that mucosal inoculation of vacciniaimmune BALB/c mice with recombinant vaccinia expressing HIV-1 gp160 induced specific serum antibody and strong HIV-1specific cytotoxic T lymphocyte responses. For live viral vaccine vectors, it is essential to evade the preexisting anti-vector immu-nity and to stimulate robust immune responses at the site of viral transmission (e.g. influenza and HIV-1). For individuals who did not receive smallpox vaccination (born after 1980), the preexisting anti-vector immunity is not an issue.

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This study was supported by the National Basic Research Program of China (973 project 2006CB504208 and sub-project 2005CB522903) (to ZC) and the Special Fund of Wuhan University to the Modern Virology Research Center. We also acknowledge partial financial supports by the NIH R01 grant HL080211-01 (to ZC).