Bladder and Bowel Dysfunction due to a Spontaneous Spinal Epidural Hematoma Without Paraplegia: A Case Report.

CASE: A 44-year-old man developed urinary retention due to a spontaneous spinal epidural hematoma (SSEH) at the cervicothoracic junction, without paraplegia. Symptoms improved with surgical treatment. CONCLUSION: SSEH is rare and causes acute neck or back pain and progressive paralysis. Patients with advanced myelopathy due to spinal cord compression lesion including SSEH often present with bladder and bowel disorders after exacerbation of quadriplegia. However, SSEH can cause predominant bladder and bowel disorders without paraplegia or quadriplegia. Physicians should consider that there can be a manifestation of myelopathy with bladder and bowel dysfunction without quadriplegia.

Did My Hand Move in a Mirror? Body Ownership Induced by the Mirror Hand Illusion.

Although the illusion that the mirror image of a hand or limb could be recognized as a part of one's body behind the mirror, the effect of adding tactile stimulation to this illusion remains unknown. We, therefore, examined how the timing of tactile stimulation affects the induction of body ownership on the mirror image. Twenty-one healthy, right-handed participants (mean age = 23.0 ± 1.0 years, no medical history of neurological and/or psychiatric disorders) were enrolled and a crossover design was adopted in this study. Participants' right and left hands were placed on the front and back sides of the mirror, respectively, then they were asked to keep looking at their right hand in the mirror. All participants experienced two experiments; one was with tactile stimulation that was synchronized with the movement of a mirror image (synchronous condition), and the other one was with tactile stimulation that was not synchronized (asynchronous condition). The qualitative degree of body ownership for the mirrored hand was evaluated by a questionnaire. Proprioceptive drift (PD), an illusory shift of the felt position of the real hand toward the mirrored hand was used for quantitative evaluation of body ownership and measured at "baseline," "immediately after stimulation," "2 min after stimulation," and "4 min after stimulation." The results of the questionnaire revealed that some items of body ownership rating were higher in the synchronous condition than in the asynchronous condition (p < 0.05). We found that PD occurred from immediately after to 4 min after stimulation in both conditions (p < 0.01) and there was no difference in the results between the conditions. From the dissociation of these results, we interpreted that body ownership could be elicited by different mechanisms depending on the task demand. Our results may contribute to the understanding of the multisensory integration mechanism of visual and tactile stimulation during mirror illusion induction.

A new serine protease family with elastase activity is produced by Streptomyces bacteria.

We found an elastolytic activity in the culture supernatant of Streptomyces sp. P-3, and the corresponding enzyme (streptomycetes elastase, SEL) was purified to apparent homogeneity from the culture supernatant. The molecular mass of purified SEL was approximately 18 kDa as judged by SDS-PAGE analysis and gel-filtration chromatography. Utilizing information from N-terminal amino acid sequencing of SEL and mass spectrometry of SEL tryptic fragments, we succeeded in cloning the gene-encoding SEL. The cloned SEL gene contains a 726 bp ORF, which encodes a 241 amino acid polypeptide containing a putative signal peptide for secretion (28 amino acid) and pro-sequence (14 amino acid). Although the deduced primary structure of SEL has sequence similarity to proteins in the S1 protease family, the amino acid sequence shares low identity (< 31.5 %) with any known elastase. SEL efficiently hydrolyses synthetic peptides having Ala or Val in the P1 position such as N-succinyl-Ala-Ala-(Pro or Val)-Ala-p-nitroanilide (pNA), whereas reported proteases by streptomycetes having elastolytic activity prefer large residues, such as Phe and Leu. Compared of kcat/Km ratios for Suc-Ala-Ala-Val-Ala-pNA and Suc-Ala-Ala-Pro-Ala-pNA with subtilisin YaB, which has high elastolytic activity, Streptomyces sp. P-3 SEL exhibits 12- and 121-fold higher, respectively. Phylogenetic analyses indicate that the predicted SEL protein, together with predicted proteins in streptomycetes, constitutes a novel group within the S1 serine protease family. These characteristics suggest that SEL-like proteins are new members of the S1 serine protease family, which display elastolytic activity.

Early stage periarticular injection during total knee arthroplasty may provide a better postoperative pain relief than late-stage periarticular injection: a randomized-controlled trial.

PURPOSE: This study was performed to determine whether periarticular injection performed in the early stage of total knee arthroplasty (TKA) could provide a better postoperative pain relief than periarticular injection performed in the late stage of TKA. The hypothesis was based on the concept that analgesic intervention before the onset of noxious stimuli would be associated with less postoperative pain. METHODS: A total of 105 participants were randomly assigned to receive superficial injection just prior to arthrotomy (early stage periarticular injection group) or superficial injection after implanting the prosthesis (late-stage periarticular injection group) in patients undergoing unilateral TKA with 1:1 treatment allocation. In both groups, deep injection was performed according to the same schedule (just prior to implanting prosthesis). The solution consisted of 300 mg of ropivacaine, 8 mg of morphine, 40 mg of methylprednisolone, 50 mg of ketoprofen, and 0.3 mg of epinephrine mixed with normal saline to a final volume of 60 mL. All surgeries were managed under general anesthesia without any regional blocks. Registry-specified primary outcome was postoperative pain score at rest measured at the recovery room using a 100-mm visual analog scale (VAS). The VAS score was compared between two groups and assessed to reach the reported threshold values for the minimal clinically important difference (MCID) of 10 mm for the postoperative VAS score. RESULTS: The VAS score at the recovery room was significantly lower in the early stage periarticular injection group than the late-stage periarticular injection group (23 ± 25 mm versus 39 ± 34 mm, respectively; 95% confidence interval 4-28 mm; p = 0.0078). The mean difference in the primary outcome fulfilled the MCID value. CONCLUSIONS: Bringing forward the timing of periarticular injection may provide significant and clinically meaningful improvement in pain following TKA under general anesthesia. LEVEL OF EVIDENCE: I.

Induced pluripotent stem cell reprogramming by integration-free Sendai virus vectors from peripheral blood of patients with craniometaphyseal dysplasia.

Studies of rare genetic bone disorders are often limited due to unavailability of tissue specimens and the lack of animal models fully replicating phenotypic features. Craniometaphyseal dysplasia (CMD) is a rare monogenic disorder characterized by hyperostosis of craniofacial bones concurrent with abnormal shape of long bones. Mutations for autosomal dominant CMD have been identified in the ANK gene (ANKH). Here we describe a simple and efficient method to reprogram adherent cells cultured from peripheral blood to human induced pluripotent stem cells (hiPSCs) from eight CMD patients and five healthy controls. Peripheral blood mononuclear cells (PBMCs) were separated from 5-7 mL of whole blood by Ficoll gradient, expanded in the presence of cytokines and transduced with Sendai virus (SeV) vectors encoding OCT3/4, SOX2, KLF4, and c-MYC. SeV vector, a cytoplasmic RNA vector, is lost from host cells after propagation for 10-13 passages. These hiPSCs express stem cell markers, have normal karyotypes, and are capable of forming embryoid bodies in vitro as well as teratomas in vivo. Further differentiation of these patient-specific iPSCs into osteoblasts and osteoclasts can provide a useful tool to study the effects CMD mutations on bone, and this approach can be applied for disease modeling of other rare genetic musculoskeletal disorders.

A novel protective MHC-I haplotype not associated with dominant Gag-specific CD8+ T-cell responses in SIVmac239 infection of Burmese rhesus macaques.

Several major histocompatibility complex class I (MHC-I) alleles are associated with lower viral loads and slower disease progression in human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections. Immune-correlates analyses in these MHC-I-related HIV/SIV controllers would lead to elucidation of the mechanism for viral control. Viral control associated with some protective MHC-I alleles is attributed to CD8+ T-cell responses targeting Gag epitopes. We have been trying to know the mechanism of SIV control in multiple groups of Burmese rhesus macaques sharing MHC-I genotypes at the haplotype level. Here, we found a protective MHC-I haplotype, 90-010-Id (D), which is not associated with dominant Gag-specific CD8+ T-cell responses. Viral loads in five D+ animals became significantly lower than those in our previous cohorts after 6 months. Most D+ animals showed predominant Nef-specific but not Gag-specific CD8+ T-cell responses after SIV challenge. Further analyses suggested two Nef-epitope-specific CD8+ T-cell responses exerting strong suppressive pressure on SIV replication. Another set of five D+ animals that received a prophylactic vaccine using a Gag-expressing Sendai virus vector showed significantly reduced viral loads compared to unvaccinated D+ animals at 3 months, suggesting rapid SIV control by Gag-specific CD8+ T-cell responses in addition to Nef-specific ones. These results present a pattern of SIV control with involvement of non-Gag antigen-specific CD8+ T-cell responses.

Immunogenicity of repeated Sendai viral vector vaccination in macaques.

Induction of durable cellular immune responses by vaccination is an important strategy for the control of persistent pathogen infection. Viral vectors are promising vaccine tools for eliciting antigen-specific T-cell responses. Repeated vaccination may contribute to durable memory T-cell induction, but anti-vector antibodies could be an obstacle to its efficacy. We previously developed a Sendai virus (SeV) vector vaccine and showed the potential of this vector for efficient T-cell induction in macaques. Here, we examined whether repeated SeV vector vaccination with short intervals can enhance antigen-specific CD8(+) T-cell responses. Four rhesus macaques possessing the MHC-I haplotype 90-120-Ia were immunized three times with intervals of three weeks. For the vaccination, we used replication-defective F-deleted SeV vectors inducing CD8(+) T-cell responses specific for simian immunodeficiency virus Gag(206-216) and Gag(241-249), which are dominant epitopes restricted by 90-120-Ia-derived MHC-I molecules. All four animals showed higher Gag(206-216)-specific and Gag(241-249)-specific CD8(+) T-cell responses after the third vaccination than those after the first vaccination, indicating enhancement of antigen-specific CD8(+) T-cell responses by the second/third SeV vector vaccination even with short intervals. These results suggest that repeated SeV vector vaccination can contribute to induction of efficient and durable T-cell responses.

Impact of vaccination on cytotoxic T lymphocyte immunodominance and cooperation against simian immunodeficiency virus replication in rhesus macaques.

Cytotoxic T lymphocyte (CTL) responses play a central role in viral suppression in human immunodeficiency virus (HIV) infections. Prophylactic vaccination resulting in effective CTL responses after viral exposure would contribute to HIV control. It is important to know how CTL memory induction by vaccination affects postexposure CTL responses. We previously showed vaccine-based control of a simian immunodeficiency virus (SIV) challenge in a group of Burmese rhesus macaques sharing a major histocompatibility complex class I haplotype. Gag(206-216) and Gag(241-249) epitope-specific CTL responses were responsible for this control. In the present study, we show the impact of individual epitope-specific CTL induction by prophylactic vaccination on postexposure CTL responses. In the acute phase after SIV challenge, dominant Gag(206-216)-specific CTL responses with delayed, naive-derived Gag(241-249)-specific CTL induction were observed in Gag(206-216) epitope-vaccinated animals with prophylactic induction of single Gag(206-216) epitope-specific CTL memory, and vice versa in Gag(241-249) epitope-vaccinated animals with single Gag(241-249) epitope-specific CTL induction. Animals with Gag(206-216)-specific CTL induction by vaccination selected for a Gag(206-216)-specific CTL escape mutation by week 5 and showed significantly less decline of plasma viral loads from week 3 to week 5 than in Gag(241-249) epitope-vaccinated animals without escape mutations. Our results present evidence indicating significant influence of prophylactic vaccination on postexposure CTL immunodominance and cooperation of vaccine antigen-specific and non-vaccine antigen-specific CTL responses, which affects virus control. These findings provide great insights into antigen design for CTL-inducing AIDS vaccines.

Intranasal Sendai viral vector vaccination is more immunogenic than intramuscular under pre-existing anti-vector antibodies.

Viral vectors are promising vaccine tools for eliciting potent cellular immune responses. Pre-existing anti-vector antibodies, however, can be an obstacle to their clinical use in humans. We previously developed a Sendai virus (SeV) vector vaccine and showed the potential of this vector for efficient CD8(+) T-cell induction in macaques. Here, we investigated the immunogenicity of SeV vector vaccination in the presence of anti-SeV antibodies. We compared antigen-specific CD8(+) T-cell responses after intranasal or intramuscular immunization with a lower dose (one-tenth of that in our previous studies) of SeV vector expressing simian immunodeficiency virus Gag antigen (SeV-Gag) between naive and pre-SeV-infected cynomolgus macaques. Intranasal SeV-Gag immunization efficiently elicited Gag-specific CD8(+) T-cell responses not only in naive but also in pre-SeV-infected animals. In contrast, intramuscular SeV-Gag immunization induced Gag-specific CD8(+) T-cell responses efficiently in naive but not in pre-SeV-infected animals. These results indicate that both intranasal and intramuscular SeV administrations are equivalently immunogenic in the absence of anti-SeV antibodies, whereas intranasal SeV vaccination is more immunogenic than intramuscular in the presence of anti-SeV antibodies. It is inferred from a recent report investigating the prevalence of anti-SeV antibodies in humans that SeV-specific neutralizing titers in more than 70% of people are no more than those at the SeV-Gag vaccination in pre-SeV-infected macaques in the present study. Taken together, this study implies the potential of intranasal SeV vector vaccination to induce CD8(+) T-cell responses even in humans, suggesting a rationale for proceeding to a vaccine clinical trial using this vector.

Dominant induction of vaccine antigen-specific cytotoxic T lymphocyte responses after simian immunodeficiency virus challenge.

Cytotoxic T lymphocyte (CTL) responses are crucial for the control of human and simian immunodeficiency virus (HIV and SIV) replication. A promising AIDS vaccine strategy is to induce CTL memory resulting in more effective CTL responses post-viral exposure compared to those in natural HIV infections. We previously developed a CTL-inducing vaccine and showed SIV control in some vaccinated rhesus macaques. These vaccine-based SIV controllers elicited vaccine antigen-specific CTL responses dominantly in the acute phase post-challenge. Here, we examined CTL responses post-challenge in those vaccinated animals that failed to control SIV replication. Unvaccinated rhesus macaques possessing the major histocompatibility complex class I haplotype 90-088-Ij dominantly elicited SIV non-Gag antigen-specific CTL responses after SIV challenge, while those induced with Gag-specific CTL memory by prophylactic vaccination failed to control SIV replication with dominant Gag-specific CTL responses in the acute phase, indicating dominant induction of vaccine antigen-specific CTL responses post-challenge even in non-controllers. Further analysis suggested that prophylactic vaccination results in dominant induction of vaccine antigen-specific CTL responses post-viral exposure but delays SIV non-vaccine antigen-specific CTL responses. These results imply a significant influence of prophylactic vaccination on CTL immunodominance post-viral exposure, providing insights into antigen design in development of a CTL-inducing AIDS vaccine.

Prevalence of specific neutralizing antibodies against Sendai virus in populations from different geographic areas: implications for AIDS vaccine development using Sendai virus vectors.

A Sendai virus (SeV) vector is being developed for delivery of an HIV immunogen. SeV is not known to cause disease in humans. Because it is genetically and antigenically related to human parainfluenza virus type 1 (hPIV-1), it is important to determine whether pre-existing hPIV-1 antibodies will affect immune responses elicited by a SeV vector-based vaccine. To quantify SeV neutralizing antibodies (NAb) in human serum, a sensitive virus neutralization assay was developed using a SeV vector encoding green fluorescent protein. Samples from 255 HIV-uninfected subjects from Africa, Europe, United States, and Japan, as well as from 12 confirmed hPIV-1-infected patients, were analyzed. SeV NAb titers did not vary significantly after serum was treated with receptor-destroying enzyme, indicating that non-specific hemagglutination inhibitors did not affect the assay sensitivity. A significant correlation was observed between hPIV-1 ELISA and SeV NAb titers. SeV NAb were detected in 92.5% subjects with a median titer of 60.6 and values ranging from 5.9- 11,324. The majority had titers < 1000 with 71.7% < 100 (< 5 considered negative). There was no significant difference in titer or prevalence by gender, age range or geographic origin. However, African males had a lower titer than non-Africans of either gender (p=0.007). Overall, the prevalence of SeV NAb is high and likely due to neutralization by cross-reactive hPIV-1 antibodies. Clinical trials will be needed to assess the influence of pre-existing SeV NAb on HIV-specific immune responses elicited by a SeV vaccine vector expressing HIV.

Cytokine-based high log-scale expansion of functional human dendritic cells from cord-blood CD34-positive cells.

Dendritic cells (DCs) play a crucial role in maintaining the immune system. Though DC-based cancer immunotherapy has been suggested as a potential treatment for various kinds of malignancies, its clinical efficacies are still insufficient in many human trials. Issues that limit the clinical efficacy of DC-based immunotherapy, as well as the difficulty of the industrial production of DCs, are largely due to the limited number of autologous DCs available from each patient. We here established a possible breakthrough, a simple cytokine-based culture method to expand the log-scale order of functional human DCs. Floating cultivation of cord-blood CD34(+) cells under an optimized cytokine cocktail led these progenitor cells to stable log-scale proliferation and to DC differentiation. The expanded DCs had typical features of conventional myeloid DCs in vitro. Therefore, the concept of DC expansion should contribute significantly to the progress of DC immunotherapy.

Cytokine-based log-scale expansion of functional murine dendritic cells.

BACKGROUND: Limitations of the clinical efficacy of dendritic cell (DC)-based immunotherapy, as well as difficulties in their industrial production, are largely related to the limited number of autologous DCs from each patient. We here established a possible breakthrough, a simple and cytokine-based culture method to realize a log-scale order of functional murine DCs (>1,000-fold), which cells were used as a model before moving to human studies. METHODOLOGY/PRINCIPAL FINDINGS: Floating cultivation of lineage-negative hematopoietic progenitors from bone marrow in an optimized cytokine cocktail (FLT3-L, IL-3, IL-6, and SCF) led to a stable log-scale proliferation of these cells, and a subsequent differentiation study using IL-4/GM-CSF revealed that 3-weeks of expansion was optimal to produce CD11b+/CD11c+ DC-like cells. The expanded DCs had typical features of conventional myeloid DCs in vitro and in vivo, including identical efficacy as tumor vaccines. CONCLUSIONS/SIGNIFICANCE: The concept of DC expansion should make a significant contribution to the progress of DC-based immunotherapy.

Evaluation of the immunogenicity of replication-competent V-knocked-out and replication-defective F-deleted Sendai virus vector-based vaccines in macaques.

Viral vectors are promising vaccine tools for eliciting antigen-specific T-cell responses. We previously showed the potential of recombinant Sendai virus (SeV) vectors to induce virus-specific T-cell responses in macaque AIDS models. Here, we have evaluated the immunogenicity of replication-competent V-knocked-out and replication-defective F-deleted SeV vectors in macaques. Intranasal replication-competent and replication-defective SeV immunizations both elicited robust systemic antigen-specific T-cell responses, whereas the responses induced by the former were more durable than those by the latter. However, even the latter-induced T-cell responses remained detectable in a local, retropharyngeal lymph node two months after the immunization. These findings are useful for establishment of a vaccine protocol using SeV vectors.

Antigen-specific T-cell induction by vaccination with a recombinant Sendai virus vector even in the presence of vector-specific neutralizing antibodies in rhesus macaques.

Recombinant viral vectors are promising vaccine tools for eliciting potent cellular immune responses against immunodeficiency virus infection, but pre-existing anti-vector antibodies can be an obstacle to their clinical use in humans. We have previously vaccinated rhesus macaques with a recombinant Sendai virus (SeV) vector twice at an interval of more than 1 year and have shown efficient antigen-specific T-cell induction by the second as well as the first vaccination. Here, we have established the method for measurement of SeV-specific neutralizing titers and have found efficient SeV-specific neutralizing antibody responses just before the second SeV vaccination in these macaques. This suggests the feasibility of inducing antigen-specific T-cell responses by SeV vaccination even in the host with pre-existing anti-SeV neutralizing antibodies.

Abrogation of AIDS vaccine-induced cytotoxic T-lymphocyte efficacy in vivo due to a change in viral epitope flanking sequences.

A current promising AIDS vaccine strategy is to elicit CD8(+) cytotoxic T lymphocyte (CTL) responses that broadly recognize highly-diversified HIVs. In our previous vaccine trial eliciting simian immunodeficiency virus (SIV) mac239 Gag-specific CTL responses, a group of Burmese rhesus macaques possessing a major histocompatibility complex haplotype 90-120-Ia have shown vaccine-based viral control against a homologous SIVmac239 challenge. Vaccine-induced Gag(206-216) epitope-specific CTL responses exerted strong selective pressure on the virus in this control. Here, we have evaluated in vivo efficacy of vaccine-induced Gag(206-216)-specific CTL responses in two 90-120-Ia-positive macaques against challenge with a heterologous SIVsmE543-3 that has the same Gag(206-216) epitope sequence with SIVmac239. Despite efficient Gag(206-216)-specific CTL induction by vaccination, both vaccinees failed to control SIVsmE543-3 replication and neither of them showed mutations within the Gag(206-216) epitope. Further analysis indicated that Gag(206-216)-specific CTLs failed to show responses against SIVsmE543-3 infection due to a change from aspartate to glutamate at Gag residue 205 immediately preceding the amino terminus of Gag(206-216) epitope. Our results suggest that even vaccine-induced CTL efficacy can be abrogated by a single amino acid change in viral epitope flanking region, underlining the influence of viral epitope flanking sequences on CTL-based AIDS vaccine efficacy.

CFTR gene transfer to human cystic fibrosis pancreatic duct cells using a Sendai virus vector.

Cystic fibrosis (CF) is a fatal inherited disease caused by the absence or dysfunction of the CF transmembrane conductance regulator (CFTR) Cl- channel. About 70% of CF patients are exocrine pancreatic insufficient due to failure of the pancreatic ducts to secrete a HCO3- -rich fluid. Our aim in this study was to investigate the potential of a recombinant Sendai virus (SeV) vector to introduce normal CFTR into human CF pancreatic duct (CFPAC-1) cells, and to assess the effect of CFTR gene transfer on the key transporters involved in HCO3- transport. Using polarized cultures of homozygous F508del CFPAC-1 cells as a model for the human CF pancreatic ductal epithelium we showed that SeV was an efficient gene transfer agent when applied to the apical membrane. The presence of functional CFTR was confirmed using iodide efflux assay. CFTR expression had no effect on cell growth, monolayer integrity, and mRNA levels for key transporters in the duct cell (pNBC, AE2, NHE2, NHE3, DRA, and PAT-1), but did upregulate the activity of apical Cl-/HCO3- and Na+/H+ exchangers (NHEs). In CFTR-corrected cells, apical Cl-/HCO3- exchange activity was further enhanced by cAMP, a key feature exhibited by normal pancreatic duct cells. The cAMP stimulated Cl-/HCO3- exchange was inhibited by dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2-DIDS), but not by a specific CFTR inhibitor, CFTR(inh)-172. Our data show that SeV vector is a potential CFTR gene transfer agent for human pancreatic duct cells and that expression of CFTR in CF cells is associated with a restoration of Cl- and HCO3- transport at the apical membrane.

[Sendai virus vector: vector development and its application to health care and biotechnology].

Sendai virus (SeV) is an enveloped virus with a nonsegmented negative-strand RNA genome and a member of the paramyxovirus family. We have developed SeV vector which has shown a high efficiently of gene transfer and expression of foreign genes to a wide range of dividing and non-dividing mammalian cells and tissues. One of the characteristics of the vector is that the genome is located exclusively in the cytoplasm of infected cells and does not go through a DNA phase; thus there is no concern about unwanted integration of foreign sequences into chromosomal DNA. Therefore, this new class of "cytoplasmic RNA vector", an RNA vector with cytoplasmic expression, is expected to be a safer and more efficient viral vector than existing vectors for application to human therapy in various fields including gene therapy and vaccination. In this review, I describe development of Sendai virus vector, its application in the field of biotechnology and clinical application aiming to treat for a large number of diseases including cancer, cardiovascular disease, infectious diseases and neurologic disorders.

Naked Sendai virus vector lacking all of the envelope-related genes: reduced cytopathogenicity and immunogenicity.

BACKGROUND: Sendai virus (SeV) is a new class of cytoplasmic RNA vector that is free from genotoxicity that infects and multiplies in most mammalian cells, and directs high-level transgene expression. We improved the vector by deleting all of the envelope-related genes from the SeV genome and thus reducing its immunogenicity. METHODS: The matrix (M), fusion (F) and hemagglutinin-neuraminidase (HN) genes-deleted SeV vector (SeV/DeltaMDeltaFDeltaHN) was recovered in a newly established packaging cell line. Then, the generated SeV/DeltaMDeltaFDeltaHN vector was characterised by comparing with single gene-deleted type SeV vectors. RESULTS: This SeV/DeltaMDeltaFDeltaHN vector carrying the green fluorescent protein gene in place of the envelope-related genes could be propagated to a titer of more than 10(8) cell infectious units/ml. This vector showed an efficient transduction capability in vitro and in vivo, and the cytopathic effect and induction of neutralizing antibody in vivo were greatly reduced compared with those of single gene-deleted type SeV vectors. No activity of neutralizing antibody or anti-HN antibody was seen when SeV/DeltaMDeltaFDeltaHN was transduced ex vivo. Additional introduction of amino acid mutations that had been identified from SeV strains causing persistent infections was also effective for the reduction of cytopathic effects. CONCLUSIONS: The deletion of genes from the SeV genome and the additional mutation are very effective for reducing both the immunogenic and cytopathic reactions to the SeV vector. These modifications are expected to improve the safety and broaden the range of clinical applications of this new class of cytoplasmic RNA vector.