Primary replication of a recombinant Sendai virus vector in macaques

Munehide Kano1, Tetsuro Matano1,5, Atsushi Kato2, Hiromi Nakamura1, Akiko Takeda1, Yuriko Suzaki3, Yasushi Ami3, Keiji Terao4 and Yoshiyuki Nagai1,6

AIDS Research Centre1, Department of Viral Diseases and Vaccine Control2 and Division of Experimental Animal Research3, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-murayama, Tokyo 208-0011, Japan
Tsukuba Primate Research Centre, National Institute of Infectious Diseases, 1 Hachimandai, Tsukuba 305-0843, Japan2
Department of Microbiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan3
Toyama Institute of Health, Nakataikou-yama 17-1, Kosugi-machi, Imizu-gun, Toyama 939-0363, Japan4

Author for correspondence: Tetsuro Matano at The University of Tokyo. Fax +81 3 5841 3374. e-mail matano{at}m.u-tokyo.ac.jp


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
An efficient antigen expression system using a recombinant Sendai virus (SeV) has been established recently and its potential to induce resistance against immunodeficiency virus infections in macaques has been shown. SeV replication has been well characterized in mice, the natural host, but not in primates, including humans. Here, primary SeV replication was investigated in macaques. After intranasal immunization with a recombinant SeV expressing simian immunodeficiency virus Gag protein, SeV-Gag, robust gag expression was observed in the nasal mucosa and much lower but significant levels of gag expression were observed in the local retropharyngeal and submandibular lymph nodes (LN). Expression peaked within a week and lasted at least up to 13 days after immunization. SeV-Gag was isolated from nasal swabs consistently at day 4 but not at all at day 13. Gag expression was undetectable in the lung as well as in remote lymphoid tissues, such as the thymus, spleen and inguinal LN, indicating that the spread of the virus was more restricted in macaques than in mice. SeV-specific T cells were detectable in SeV-immunized macaques at day 7. Finally, no naive macaques showed significant levels of anti-SeV antibodies in the plasma, even after living in a cage together with an acutely SeV-infected macaque for 5 weeks, indicating that SeV transmission from SeV-infected macaques to naive ones was inefficient. None of the SeV-immunized macaques displayed appreciable clinical manifestations. These results support the idea that this system may be used safely in primates, including humans.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Virus-specific T cell responses have been shown to play an important role in controlling human immunodeficiency virus type 1 (HIV-1) infections (Ogg et al., 1998 ; Rowland-Jones et al., 1998 ; Brander & Walker, 1999 ; Seder & Hill, 2000 ). Furthermore, in macaque AIDS models, the importance of CD8+ T cells in the control of immunodeficiency virus infections has been shown by CD8+ T cell depletion using anti-CD8 antibodies in vivo (Matano et al., 1998 ; Schmitz et al., 1999 ; Jin et al., 1999 ). Thus, induction of virus-specific T cell responses would be of value for the protection against AIDS. Virus vector-based gene delivery is a promising AIDS vaccine strategy because of its potential for inducing cellular immune responses.

Sendai virus (SeV) is an enveloped virus with a negative-sense RNA genome of about 15·3 kb. It causes fatal pneumonia in mice, the natural host, but is thought to be nonpathogenic in humans (Nagai, 1999 ). We previously established a system to rescue SeV from cDNA, which enabled us to engineer the SeV genome at will and use the virus as a vector to deliver foreign genes of interest (Hasan et al., 1997 ; Yu et al., 1997 ; Nagai & Kato, 1999 ). Recent studies using animals such as mice and ferrets have shown an extremely high efficiency of SeV vector-based gene transfer in vivo (Yonemitsu et al., 2000 ). Because SeV replication requires a proteinase that is localized in the airway epithelium for Env protein processing (Nagai, 1993 ), the intranasal route would be the best for efficient delivery and this may be favourable for the induction of mucosal immune responses.

We previously knocked out the SeV V gene, an accessory gene, to obtain an attenuated SeV, V-SeV (Kato et al., 1997a , b ). V-SeV infection is not fatal in mice and the virus retains the ability to induce efficient gene transfer. Indeed, expression levels of HIV-1 Env gp120 from recombinant V-SeV-infected cells reached 6 µg per 106 cells, the highest among the vector systems available in mammalian cells (Yu et al., 1997 ).

We then constructed a recombinant V-SeV expressing the simian immunodeficiency virus (SIV) Gag protein, SeV-Gag, to evaluate the protective efficacy of its immunization in macaque AIDS models. In two sets of macaque experiments, intranasal SeV-Gag immunization elicited resistance against a pathogenic SIV, strain mac239 (SIVmac239), and a pathogenic SIV/HIV strain, SHIV89.6PD (Kano et al., 2000 ; Matano et al., 2001 ). The SeV-Gag-immunized macaques failed to control primary viraemia but greatly reduced the set-point plasma virus loads after challenge. These results prompted us to determine the pattern of SeV replication in nonhuman primates for utilizing this vector as a tool for an AIDS vaccine in humans effectively and safely. In this study, we examined primary SeV replication and SeV-specific immune responses in macaques following intranasal SeV immunization.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Preparation of recombinant SeV.
By using a safer V gene knock-out version of SeV, V-SeV (Kato et al., 1997a , b ), we constructed a recombinant SeV vector expressing SIV Gag, SeV-Gag. The plasmid pSeV(+)18bV(-), which contains the full-length genome sequence of attenuated V gene-defective SeV cDNA, was described before (Kato et al., 1996; Hasan et al., 1997 ). A gene fragment encoding SIVmac239 Gag (nt 1306–2845, GenBank accession no. M33262) (Kestler et al., 1990 ) was prepared by PCR and introduced into pSeV(+)18bV(-) to obtain pSeV(+)18bV(-)/SIVgag (Fig. 1a). Primers used for the PCR were 5' AAGCGGCCGCGAGATGGGCGTGAGAAACTCCG and 5' TTGC G G C C G C G ATGAACTTTCACCCTAAGTTTTTCTTACT G T G A C-TACTGGTCTCCTCCAAAG. The gag gene fragment was inserted into the NotI site, immediately upstream of the N-terminal coding region of pSeV(+)18bV(-). SeV-control and SeV-Gag were recovered from pSeV(+)18bV(-) and pSeV(+)18bV(-)/SIVgag, respectively. A recombinant SeV expressing HIV-1 gp120 was obtained as described previously (Yu et al., 1997 ). The titre of SeV, measured in cell infectious units (CIU)/ml was assayed on CV-1 cells by immunostaining using an anti-SeV antibody, as described before (Kiyotani et al., 1990 ).



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Fig. 1. SIV Gag expression in SeV-Gag-infected cells. (a) Structure of pSeV(+)18bV(-)/SIVgag, a plasmid generating an antigenomic RNA of SeV-Gag. (b) Western blot analysis using a monoclonal mouse anti-p27 antibody. CV-1 cells were lysed 24 h after mock infection (lane 1), SeV-control infection (lane 2) or SeV-Gag infection (lane 3). (c) Replication kinetics of SeV-control ({circ}) and SeV-Gag ({bullet}) in CV-1 cells under multiple cycle growth conditions in the presence of trypsin (7·5 µg/ml) (Sakai et al., 1999). At the initial infection, cells were infected with SeV-control or SeV-Gag at an m.o.i. of 0·05. SeV titres in the supernatants harvested at several time-points were assayed on CV-1 cells by immunostaining. (d) Dot plots showing Gag-specific intracellular IFN-{gamma} induction in PBMCs after coculture with SeV-Gag-infected cells. PBMCs from an SIV-infected macaque were cocultured with the autologous B-LCLs infected with SeV-control (left panel), SeV-Gag (middle panel) or Vv-Gag (right panel). Dot plots gated on CD3+CD8+ lymphocytes are shown. The per cent of IFN- {gamma}+ cell number to the gated cell number in each dot plot is shown.

 
{blacksquare} Western blot analysis.
CV-1 cells were seeded at a density of 4x105 cells per well in a 6-well plate, grown overnight and then infected with SeV-control or SeV-Gag at an m.o.i. of 5. A day later, the cells were lysed with 600 µl of triple-detergent lysis buffer (50 mM Tris–HCl, pH 8·0, 150 mM NaCl, 0·02% sodium azide, 0·1% SDS, 0·5% sodium deoxycholate, 0·1 mg/ml PMSF and 1% Triton X-100). Western blot analysis using a monoclonal mouse anti-p27 (SIV Gag CA) antibody was performed as described before (Matano et al., 1993 ). For each lane, 10 µl of cell lysate was loaded for analysis.

{blacksquare} Animals.
Cynomolgus macaques (Macaca fascicularis) and rhesus macaques (Macaca mulatta) used in this study were maintained in accordance with the institutional guidelines for laboratory animals. These macaques tested negative for SeV and SIV before use. Monkey R010 received a control DNA vaccine, 800 µg pCMVN DNA (Matano et al., 2000 ) by intramuscular inoculation and 10 µg pCMVN DNA by gene gun (four times), from 12 to 6 weeks before the SeV-control immunization for another experiment. Blood collections, sampling of nasal swabs and immunizations were performed under ketamine anaesthesia.

{blacksquare} SeV-Gag RNA expression in tissues.
Cells from the lymph nodes (LN), thymus and spleen were prepared by mincing the tissues. Cells from the nasal mucosa and lung were prepared after treatment with collagenase type I and dispase (Life Technologies). These cells were washed with PBS three times before RNA extraction. Peripheral blood mononuclear cells (PBMCs) were prepared from whole blood samples using Ficoll–Paque Plus (Amersham-Pharmacia). RNA was extracted from cells using an RNA Extraction kit from Qiagen, according to the manufacturer’s instructions. Nested RT–PCR was performed using SIV gag-specific primers (5' AGAAACTCCGTCTTGTCAGG and 5' TGATAATCTGCATAGCCGC for the first RT–PCR and 5' GATTAGCAGAAAGCCTGTTGG and 5' TGCAACCTTCTGACAGTGC for the second PCR). The levels of gag RNA were quantified by limiting dilution of RNA samples to determine the end-point, as described before (Shibata et al., 1997 ).

{blacksquare} SeV recovery from nasal swabs or nasal mucosa.
The nasal swab sample diluted in RPMI-1640 (Life Technologies) was injected into the allantoic cavity of chickens’ eggs for recovery of SeV. In case of SeV recovery from the cells prepared from the nasal mucosa, the cells were freeze-thawed twice and 105 cells suspended in RPMI-1640 were injected into the allantoic cavity of chickens’ eggs. After 72 h of incubation, the allantoic fluid was harvested and subjected to a haemagglutination (HA) assay to detect SeV, as described before (Kato et al., 1996 ).

{blacksquare} Antibody ELISA.
An ELISA to measure plasma levels of anti-SeV antibodies was performed using inactivated SeV (Watanabe et al., 2000 ).

{blacksquare} Flow cytometry analysis of antigen-specific interferon-{gamma} (IFN-{gamma}) induction.
Autologous herpesvirus papio-immortalized B lymphoblastoid cell lines (B-LCLs) (Voss et al., 1992 ) were infected with a control vaccinia virus (VV) vector (Vv-control) (Mackett et al., 1982 ), SeV-control, a recombinant VV vector expressing SIV Gag (Vv-Gag) or SeV-Gag at an m.o.i. of 5 and, 1 day later, used as a nonspecific control, an SeV-specific, a Gag-specific or an SeV-Gag-specific stimulator. For stimulation, 106 PBMCs were cocultured with 105 stimulator cells in the presence of GolgiStop (monensin) (Pharmingen) for 6 h. Then, intracellular IFN-{gamma} staining was performed using Cytofix-Cytoperm kit (Pharmingen), according to the manufacturer's protocol, as described before (Matano et al., 2001 ).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Gag expression from SeV-Gag in vitro
SeV-Gag was rescued from the plasmid pSeV(+)18bV(-)/SIVgag (Fig. 1a). Expression of unprocessed SIV Gag (p55) in SeV-Gag-infected CV-1 cells was confirmed by Western blot analysis (Fig. 1b) as well as by immunostaining (data not shown) using a monoclonal anti-SIV Gag p27 antibody. The recombinant virus showed slightly slower proliferation rate compared with that of SeV-control (Fig. 1c). This retarded replication might be due to the increase in genome length (by 1·6 kb) as a result of the insertion [pSeV(+)18bV(-), 15·4 kb; pSeV(+)18bV(-)/SIVgag, 17·0 kb] of the Gag protein-coding region (Hasan et al., 1997 ; Yu et al., 1997; Sakai et al., 1999 ).

Restimulation of Gag-specific CD8+ T cells by SeV-Gag-infected cells in vitro
Using flow cytometry analysis of antigen-specific intracellular IFN-{gamma} induction (Lavini et al., 1997 ; Butz & Bevan, 1998 ; Murali-Krishna et al., 1998 ; Donahoe et al., 2000 ; Gea-Banacloche et al., 2000 ), we examined if SeV-Gag-infected cells can really restimulate Gag-specific CD8+ T cells in vitro (Fig. 1d). We used PBMCs derived from a rhesus macaque chronically infected with SIV as a source of Gag-specific CD8+ T cells. This animal received a proviral DNA vaccine, followed by SIVmac239 challenge, as described before (Matano et al., 2000 ).

Coculture of PBMCs with autologous B-LCLs infected with recombinant VV expressing SIV Gag (Vv-Gag) showed SIV Gag-specific intracellular IFN-{gamma} induction in CD8+ T cells. Similar frequencies of CD8+IFN-{gamma}+ T cells were observed in PBMCs cocultured with SeV-Gag-infected B-LCLs. No significant induction of CD8+IFN-{gamma}+ T cells was found in the PBMCs cocultured with SeV-control-infected B-LCLs. These results indicate that the SeV-Gag-infected B-LCLs restimulated Gag-specific CD8+ T cells efficiently, confirming the potential of our SeV vector system for inducing specific T cell responses.

SeV-Gag expression in the nasal mucosa after its intranasal immunization into macaques
The first in vivo experiment was performed to examine the primary replication of SeV-Gag in macaques. Six cynomolgus macaques were inoculated intranasally with 108 CIU SeV-Gag. None of the monkeys showed apparent clinical symptoms. Two of them (C3880 and C4325) were euthanized at day 4, two (C3993 and C4240) at day 7 and two (C3882 and C4324) at day 13 after the inoculation, respectively. Cells were prepared from each tissue taken at autopsy and RNA was extracted from the cells. Histological analysis showed only a slight inflammatory change in the nasal mucosa but no pathological change in other tissues, including the lung (data not shown).

In the cells prepared from the nasal mucosa, quantitative RT–PCR detected 1·7x104 or 2·6x105 copies of gag RNA per 106 cells at day 4 (Fig. 2a). Expression levels were less prominent but still significant at days 7 and 13. These results indicate efficient SeV-Gag expression in the nasal mucosa after intranasal SeV-Gag inoculation.



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Fig. 2. Levels of SIV gag RNA in macaques after intranasal SeV-Gag immunization. Levels of SIV gag RNA in cells prepared from the nasal mucosa (a), retropharyngeal LN (b) and submandibular LN (c) are shown. RNA levels in C3880 or C4325 at day 4 were not determined because we failed to obtain the submandibular LN from these monkeys.

 
Then, we examined if SeV was recovered from the nasal swab. As shown in Table 1, the virus was recovered from the swab at day 4 in all six animals. At day 7, virus was recovered in two of four macaques, both of which showed higher levels of gag RNA in the nasal mucosa. At day 13, no virus was recovered in the animals. We also examined if the virus was recovered from 105 cells prepared from the nasal mucosa (Table 1). The virus was recovered from the samples obtained at day 4 but not from those at days 7 or 13. These results indicate that SeV-Gag replication rates reached the peak within a week after inoculation.


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Table 1. SeV-Gag replication in macaques after intranasal inoculation

 
SeV-Gag expression in the primary LN of the nasal cavity after the immunization
The retropharyngeal LN and the submandibular LN receive the primary lymphocyte drainage from the nasal cavity (Suen & Stern, 1996 ) and we found gag RNA in both of them (Fig. 2b, c). The levels of gag RNA were about 1/20 of those in the nasal mucosa in each individual animal at days 7 and 13. At day 4, however, the level of gag RNA in the retropharyngeal LN was less than 1/50 of that in the nasal mucosa in each individual animal. Thus, no significant difference in the levels of gag RNA in the LN was observed between days 4 and 7, although the level of RNA in the nasal mucosa at day 4 was significantly higher than that at day 7.

SeV-Gag expression in other tissues after immunization
We also examined the expression of gag in the lung, thymus, spleen and inguinal LN but found no expression in any of them (Table 1).

SeV-specific immune responses in immunized macaques
The second in vivo experiment was performed to examine SeV-specific immune responses in macaques after intranasal SeV immunization. Two rhesus macaques (R010 and R014) received 108 CIU SeV-control and three (R013, R015 and R017) received 108 CIU SeV-Gag. The former group showed no IFN-{gamma} induction after Gag-specific stimulation, while the latter group showed significant levels of Gag-specific T cell responses (Matano et al., 2001 ). None of them showed apparent clinical symptoms after immunization.

Then, we examined SeV-specific T cell responses by flow cytometry analysis of antigen-specific intracellular IFN-{gamma} induction. SeV-specific T cell responses were induced quickly in all animals after immunization (Fig. 3). Significant levels of SeV-specific T cells were detected in PBMCs at week 1. Especially, efficient induction of specific CD4 T cells was observed. At week 3, all animals showed higher frequencies of SeV-specific CD8+ T cells.



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Fig. 3. Dot plots showing SeV-specific IFN-{gamma} induction in peripheral CD4+ and CD8+ T cells in the immunized macaques. PBMCs at week 1 (a) or week 3 (b) were cocultured with B-LCLs infected with SeV-control and subjected to flow cytometry. Dot plots gated on CD3+CD4+ (upper panel in each) or CD3+CD8+ (lower panel in each) lymphocytes are shown. The per cent of IFN-{gamma}+ cell number to the gated cell number in each dot plot is shown. The per cent of IFN-{gamma}+ cell number after nonspecific stimulation (coculture with Vv-control-infected B-LCLs) is shown in parentheses. ND, Not determined.

 
In all of them, plasma anti-SeV antibody was undetectable at week 1 but significant levels of the antibody appeared at week 2 after immunization (Fig. 4).



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Fig. 4. Plasma anti-SeV antibody levels detected by ELISA in SeV-immunized macaques. The absorbance obtained with plasma diluted by 1:1000 is shown. The cut-off line is shown by a dotted line.

 
SeV transmission between macaques
The third in vivo experiment was performed to examine if SeV can be transmitted from SeV-immunized macaques to naive ones. In this experiment, we used recombinant SeV expressing HIV-1 gp120 (Yu et al., 1997 ). Two pairs of cynomolgus macaques, C99001 and C99002, and C99005 and C99003, were used. In each pair, one macaque (C99001 and C99005, respectively) was immunized intranasally with 2x108 CIU SeV and 18 h later was put into a single cage together with its partner (C99002 and C99003, respectively). Each pair of monkeys was maintained together for 5 weeks. None of them showed apparent clinical symptoms after immunization. Due to the limitation of the cage size, small macaques that were 8 months old were chosen for this experiment.

We measured plasma anti-SeV antibody levels to examine the possibility of SeV transmission (Fig. 5). Similar to the second in vivo experiment, plasma anti-SeV antibodies were clearly detectable at week 2 in both of the SeV-immunized macaques. In contrast, none of the naive macaques showed significant levels of anti-SeV antibodies. These results indicate inefficient SeV transmission from SeV-infected macaques to naive ones.



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Fig. 5. Plasma anti-SeV antibody levels detected by ELISA in naive and SeV-immunized macaques being maintained together. C99002 was in a cage together with SeV-immunized C99001, and C99003 with SeV-immunized C99005. The absorbance obtained with plasma diluted by 1:1000 is shown.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
We previously reported the potential of our SeV-Gag vaccine system for inducing cellular immune responses efficiently and its protective efficacy against SIV and SHIV infections in macaque AIDS models (Kano et al., 2000 ; Matano et al., 2001). In this study, we focused on the primary replication of the SeV vector in macaques.

In macaque experiments, SIV gag RNA was detected dominantly in the nasal mucosa and its local LN after intranasal SeV-Gag immunization. The detected RNA consists of the genomic RNA and the mRNA but we confirmed mRNA expression by quantitative RT–PCR using primers that discriminate SeV mRNA from SeV genomic RNA (Kato et al., 2001 ) (data not shown). Efficient gag expression in the local LN as well as the nasal mucosa after immunization suggests its potential for induction of mucosal as well as systemic immune responses.

None of the monkeys in either the previous studies (Hurwitz et al., 1997 ; Kano et al., 2000 ) or the present study showed significant pathological symptoms after intranasal SeV immunization. SeV-Gag expression was localized in the nasal mucosa and its local LN and its expression levels reached the peak within a week after immunization. Such tissue restriction is compatible with its replication pattern in mice, which is localized in the airway epithelium. In contrast to the case in mice, we found neither pathological changes nor SeV-Gag expression in the lung in macaques, indicating that the spread of the virus was more restricted in macaques than that in mice.

In general, the proliferation of SeV-control is not slower than that of recombinant SeVs carrying inserted genes in vitro. However, no clinical symptoms were observed even in the SeV-control-immunized macaques. Furthermore, we found no significant differences in the efficiency of SeV recovery from the nasal swabs between SeV-control- and SeV-Gag-immunized macaques (data not shown), indicating that even SeV-control can be well controlled in macaques. Therefore, the SeV vector system could be used safely for the expression of various kinds of antigens in primates.

An intranasal SeV-Gag immunization into macaques induced anti-SeV antibodies and SeV-specific T cells quickly. Especially, all immunized macaques showed SeV-specific T cells at week 1, suggesting the potential of the specific T cells for controlling SeV replication. In spite of these responses specific to the virus vector itself, significant levels of Gag-specific T cell induction were also detected (Matano et al., 2001 ). We found no relationship between SeV-specific and Gag-specific immune responses.

One of the characteristics of the SeV vector system is that the inserted gene is expressed more promptly and efficiently than any other SeV-specific genes derived from the vector because its accommodation is close to the 3' terminus of the SeV genome (Nagai, 1999 ). In addition, the inserted gene fragment has been shown to be stable in the vector both in vitro (Yu et al., 1997 ) and in vivo (in mice) (Akaike et al., 2000 ). We confirmed expression of Gag as well as SeV in the cells infected with the recovered virus from the swabs in the SeV-Gag-immunized macaques used in our previous experiment (Kano et al., 2000), indicating that the recovered vector still expressed the antigen, Gag. These characteristics of the SeV vector system may contribute to the efficient induction of immune responses specific to the target antigen in the presence of strong immune responses specific to the virus vector itself.

In the third in vivo experiment, SeV transmission from acutely SeV-infected macaques to naive ones was not detected but a little increase in anti-SeV antibody levels below the cut-off line was observed. Thus, the possibility of its transmission was not excluded completely. However, our results indicate that SeV transmission between macaques was inefficient.

In summary, we first analysed primary SeV replication and immune responses in macaques after intranasal immunization with a recombinant SeV. SeV replication was localized around the nasal mucosa and was well controlled. Furthermore, SeV transmission from SeV-immunized macaques to others was inefficient. These results may support the idea that our system can be safely used in primates, including humans. Also, a safer, replication-defective SeV vector lacking F gene is available now (Li et al., 2000 ). In combination with our previous results, the present study indicates that the SeV vector system can induce effective cellular immune responses against pathogenic virus infections, while it is well controlled by strong immune responses against the vector itself in primates. Thus, this system may be a promising, safe and effective tool for vaccines against infections with various kinds of micro-organisms.


   Acknowledgments
 
We thank B. Moss for giving us a vaccinia virus vector; A. Kojima and T. Sata for giving us an anti-p27 antibody and their help; F. Ono, K. Komatsuzaki, K. Oto, K. Mori, R. Mukai and A. Yamada for assistance in the animal experiments; and K. Nibu, T. Shioda and H. Yoshikura for their help and suggestions. This work was supported by grants for AIDS Research and for Gene Therapy from the Ministry of Health and Welfare, the Ministry of Education, Culture, Sports, Science and Technology, and Health Sciences Foundation in Japan.


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Discussion
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Received 11 December 2001; accepted 5 March 2002.