Anti-IL-6 receptor antibody causes less promotion of tuberculosis infection than anti-TNF-α antibody in mice.

Objective. Our aim was to investigate the effects of IL-6 blockade on the progression of Mycobacterium tuberculosis (TB) and compare them with those of TNF-α blockade in mice. Methods. Mice were intravenously infected with TB and injected with antibodies. Survival was monitored and histological and immunological studies were carried out. Results. All anti-IL-6R Ab-treated mice and 8 of 10 control mice survived until sacrificed 224 days after TB challenge, whereas anti-TNF-α Ab-treated mice all died between 120 and 181 days. Anti-IL-6R Ab-treated mice exhibited no significant differences in TB CFU in organs, including the lungs, and no deterioration in histopathology compared to control mice at 4 weeks. In contrast, anti-TNF-α Ab-treated mice exhibited increased TB CFU and greater progression of histopathological findings in organs than control mice. Spleen cells from anti-TNF-α Ab-treated mice had decreased antigen-specific response in IFN-γ release and proliferation assays. The results in anti-IL-6R Ab-treated mice suggest that spleen cell responses were decreased to a lesser degree. Similar results were obtained in IL-6 knockout (KO) mice, compared with TNF receptor 1 (TNFR1) KO and TNFR1/IL-6 double KO (DKO) mice. Conclusion. IL-6R blockade promotes the progression of TB infection in mice far less than TNF-α blockade.

Novel prophylactic vaccine using a prime-boost method and hemagglutinating virus of Japan-envelope against tuberculosis.

OBJECTIVE: Mycobacterium tuberculosis infection is a major global threat to human health. The only tuberculosis (TB) vaccine currently available is bacillus Calmette-Guérin (BCG), although it has no efficacy in adults. Therefore, the development of a novel vaccine against TB for adults is desired. METHOD: A novel TB vaccine expressing mycobacterial heat shock protein 65 (HSP65) and interleukin-12 (IL-12) delivered by the hemagglutinating virus of Japan- (HVJ)- envelope was evaluated against TB infection in mice. Bacterial load reductions and histopathological assessments were used to determine efficacy. RESULTS: Vaccination by BCG prime with IgHSP65+murine IL-12/HVJ-envelope boost resulted in significant protective efficacy (>10, 000-fold versus BCG alone) against TB infection in the lungs of mice. In addition to bacterial loads, significant protective efficacy was demonstrated by histopathological analysis of the lungs. Furthermore, the vaccine increased the number of T cells secreting IFN-γ. CONCLUSION: This vaccine showed extremely significant protection against TB in a mouse model, consistent with results from a similar paper on cynomolgus monkeys. The results suggest that further development of the vaccine for eventual testing in clinical trials may be warranted.

Development of therapeutic and prophylactic vaccine against Tuberculosis using monkey and transgenic mice models.

PURPOSE: BCG is not efficacious against M. tuberculosis (TB) in adult. Therefore, novel TB vaccines were established by using three kinds of animal models (cynomolgus monkey model which is the best animal model of human TB, IL-2R knock out SCID mice as a human immune model, and granulysin transgenic mouse). METHODS AND RESULTS: DNA vaccine expressing TB Hsp65 and IL-12 was delivered by the hemagglutinating virus of Japan (HVJ)-envelope. The BCG prime followed by Hsp65+IL-12/HVJ vaccine boost showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, 33% of monkeys were alive in BCG alone group. Furthermore, the prolongation of survival period of the monkey was observed by the combination of BCG and DNA vaccine even when the boost was performed after long-term period (4month) from prime. This combination also improved the erythrocyte sedimentation rate (ESR), increased the body weight, and augmented the proliferation of PBL and IL-12 production at higher levels than BCG alone or saline. Furthermore, this vaccine exerted therapeutic efficacy in IL-2R knock out SCID-PBL/hu mice, which were transplanted with human T cells. Granulysin is an important defensive molecule expressed by human T cells and NK cells and has a cytolytic activity against microbes including Mycobacterium tuberculosis (TB) and tumors. Expression of 15kD (15K) granulysin protein and mRNA in CD8 positive T cells in the patients infected with drug sensitive (TB) or multi-drug resistant (MDR-TB) M. tuberculosis were lower than that in the healthy volunteers, suggesting that granulysin treatment might improve the tuberculous disease in human. Therefore, we established two kinds of granulysin transgenic mice (15K granulysin transgenic mice and 9K granulysin transgenic mice). It was demonstrated that 15K granulysin transgenic mice as well as 9K granulysin transgenic mice exerted in vivo anti-TB effect, including the decrease of the number of TB and augmentation of the CTL activity. These are the first findings which demonstrate in vivo effects of 15K granulysin and 9K granulysin against TB infection. Moreover, DNA vaccine expressing 15K granulysin showed a therapeutic activity against TB in mice. CONCLUSION: These data indicate that monkey, IL-2R gene-knock out SCID-PBL/hu and granulysin transgenic mice models provide useful tools for the development of novel vaccines (HVJ-Envelope/Hsp65 DNA + IL-12 DNA vaccine and granulysin vaccine) against TB.

Novel therapeutic vaccine: granulysin and new DNA vaccine against Tuberculosis.

PURPOSE: Multi-drug resistant (MDR) Mycobacterium Tuberculosis (M.TB) is a big problem in the world. We have developed novel TB therapeutic vaccines. METHODS AND RESULTS: DNA vaccine expressing mycobacterial heat shock protein 65 and IL-12 was delivered by the hemagglutinating virus of Japan (HVJ)-envelope. M. TB, MDR-TB or extremenly drug resistant (XDR-TB) was injected i.v. into DBA/1 mice, and treated with the vaccine three times. This HVJ-E/Hsp65DNA+IL-12DNA vaccine provided strong therapeutic efficacy against MDR-TB and XDR-TB (prolongation of survival time and the decrease in the number of TB) in mice. Therapeutic effect of this vaccine on TB infection was also demonstrated in chronic TB infection murine model using aerosol infection intratracheally. On the other hand, granulysin protein produced from CTL has lethal activity against TB. Granulysin protein vaccine also exerted strong therapeutic effect. Furthermore, we extended our studies to monkey model, which is currently the best animal model of human TB. Hsp65DNA+IL-12 DNA vaccine exerted strong therapeutic efficacy (100% survival and augmentation of immune responses) in the TB-infected monkeys. In contrast, the survival of the saline control group was 60% at 16 week post-challenge. HVJ-Envelope/HSP65 DNA+IL-12 DNA vaccine increased the body weight of TB-infected monkeys, improved the erythrocyte sedimentation rate, and augmentated the immune responses (proliferation of PBL and IL-2 production). The enhancement of IL-2 production from monkeys treated with this vaccine was correlated with the therapeutic efficacy of the vaccine. CONCLUSION: These data indicate that novel vaccines might be useful against TB including XDR-TB and MDR-TB for human therapeutic clinical trials.

Tuberculosis vaccine development: The development of novel (preclinical) DNA vaccine.

A third of the world's population is infected with Mycobacterium tuberculosis, and 2 million people die from tuberculosis every year. The only tuberculosis vaccine currently available is an attenuated strain of Mycobacterium bovis BCG, although its efficacy against adult tuberculosis disease remains controversial. Furthermore multi-drug resistant tuberculosis is becoming big problems in the world. Therefore, the development of novel therapeutic vaccine as well as novel prophylactic vaccine against tuberculosis is required. This review provides a summary of novel vaccines (especially DNA vaccines) in preclinical stage using mouse, guinea pig and monkey models. In several promising novel vaccines, the studies were extended to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. The review also provides recent advances of the precise studies of induction of immunity including CD8 positive cytotoxic T cells and effector molecules such as granulysin by these vaccines, against multi-drug resistant tuberculosis and extremely drug resistant tuberculosis.

[Anti-tuberculosis immunity by cytotoxic T cells * granulysin and the development of novel vaccines (HSP-65 DNA+IL-12 DNA)].

CDC and ACET in U.S.A. reported that novel vaccines instead of BCG are required for the protection against infection of Mycobacterium tuberculosis worldwide. However, no novel vaccine for clinical use has not yet been developed in the world including U.S.A. and Europe. We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-envelope and -liposome (HSP65+IL-12/HVJ). This vaccine provided remarkable protective efficacy in mouse compared to the BCG vaccine on the basis of C.F.U of number of TB, survival, an induction of the CD8 positive CTL activity and improvement of the histopathological tuberculosis lesions. This vaccine also provided therapeutic efficacy against multidrug resistant TB (MDR-TB) and extremely drug resistant TB (XDR-TB) in murine models. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses. Furthermore, the BCG priming and HSP65+IL-12/HVJ vaccine (booster) by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). Furthermore, this vaccine exerted therapeutic efficacy (100% survival) and augmentation of immune responses in the TB-infected monkeys. These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical trials. The review also provides recent advances of the precise studies of induction of immunity including CD8 positive cytotoxic T cells and effector molecules such as granulysin by these vaccines, against multi-drug resistant tuberculosis and extremely drug resistant tuberculosis.

Effects of Fatty Acid Therapy in Addition to Strong Statin on Coronary Plaques in Acute Coronary Syndrome: An Optical Coherence Tomography Study.

BACKGROUND Vascular healing response associated with adjunctive n-3 polyunsaturated fatty acid therapy therapy in patients receiving strong statin therapy remains unclear. The aim of this study was to evaluate the effect of polyunsaturated fatty acid therapy with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) in addition to strong statin therapy on coronary atherosclerotic plaques using optical coherence tomography. METHODS AND RESULTS This prospective multicenter randomized controlled trial included 130 patients with acute coronary syndrome treated with strong statins. They were assigned to either statin only (control group, n=42), statin+high-dose EPA (1800 mg/day) (EPA group, n=40), statin+EPA (930 mg/day)+DHA (750 mg/day) (EPA+DHA group, n=48). Optical coherence tomography was performed at baseline and at the 8-month follow-up. The target for optical coherence tomography analysis was a nonculprit lesion with a lipid plaque. Between baseline and the 8-month follow-up, fibrous cap thickness (FCT) significantly increased in all 3 groups. There were no significant differences in the percent change for minimum FCT between the EPA or EPA+DHA group and the control group. In patients with FCT <120 µm (median value), the percent change for minimum FCT was significantly higher in the EPA or EPA+DHA group compared with the control group. CONCLUSIONS EPA or EPA+DHA therapy in addition to strong statin therapy did not significantly increase FCT in nonculprit plaques compared with strong statin therapy alone, but significantly increased FCT in patients with thinner FCT. Registration URL: https://www.umin.ac.jp/ctr/; Unique identifier: UMIN 000012825.

Preclinical study and clinical trial of a novel therapeutic vaccine against multi-drug resistant tuberculosis.

[Purpose] Multi-drug resistant (MDR), Mycobacterium tuberculosis (TB) is a big problem in the world. We have developed novel TB therapeutic vaccine (HVJ-E/HSP65 DNA +IL-12 DNA). [Methods and Results] DNA vaccine expressing TB heat shock protein 65 and IL-12 was delivered by the hemagglutinating virus of Japan (HVJ)-envelope. This vaccine provided remarkable protective efficacy and strong therapeutic efficacy against MDR-TB and XDR-TB in murine models. Furthermore, this vaccine provided therapeutic efficacy of prolongation of survival time of TB infected monkeys and augmented the immune responses. Therefore, the preclinical tests were studied for clinical trial. The injection of 100 µg of the vaccine /mouse i.m. three times in two weeks induced significantly strong production of IFN-γ and IL-2. 100 µg and 200 µg DNA vaccine/mouse i.m. augmented the production of these cytokines compared with 25 µg DNA vaccine/mouse i.m.. The ratio of 100 µg pDNA to 1AU HVJ-E enhanced the production of IFN-γ and IL-2. The decrease in the number of M. tuberculosis in liver of mice was observed by the vaccination of 100µg pDNA. By using these conditions, safety pharmacology study and toxicology test is being studied in monkeys administered by GMP level DNA vaccines. By the toxicology test using monkeys, high dose GMP level vaccine/ monkey is administrated. Safety pharmacological study of repeated administration is also being investigated in GLP level. Furthermore, we have planned to do clinical phase I trial. Targets are human patients with MDR-TB. The safety and tolerability of the vaccine will be evaluated. [Conclusion and recommendations] These data indicate that our novel vaccine might be useful against tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical applications.

Development of vaccines and passive immunotherapy against SARS coronavirus using mouse and SCID-PBL/hu mouse models.

We have investigated novel vaccines strategies against severe acute respiratory syndrome (SARS) CoV infection using cDNA constructs encoding the structural antigens; spike (S), membrane (M), envelope (E), or nucleocapsid (N) protein, derived from SARS CoV (strain HKU39849, TW1, or FFM-1). As SARS-CoV is thought to infect the alveolar epithelial cell of the lung,in the present study, a type II alveolar epithelial cell clone, T7, was used to analyze the mechanism of CTL against SARS CoV membrane antigens. Mice vaccinated with SARS CoV (N) DNA or (M) DNA using pcDNA 3.1 (+) plasmid vector showed T-cell immune responses (CTL induction and proliferation) against type II alveolar epithelial cells (T7) transfected with SARS (N) or (M) DNA, respectively. To determine whether these DNA vaccines could induce T-cell immune responses in humans as well as in mice, SCID-PBL/hu mice were immunized with these DNA vaccines. PBL from healthy human volunteers were administered i.p. into IL-2 receptor gamma-chain-disrupted NOD-SCID mice [IL-2R(-/-) NOD-SCID]. SCID-PBL/hu mice thus constructed can be used to analyze the human immune response in vivo. The SCID-PBL/hu mice were immunized with SARS (N) DNA or (M) DNA and analyzed for a human T-cell immune response. The M DNA vaccine enhanced CTL activity and proliferation in the presence of M peptide in SCID-PBL/hu mice. Furthermore, the SARS N DNA vaccine induced CTL activity (IFN-gamma production by recombinant N protein or N protein-pulsed autologous B blast cells) and proliferation of spleen cells in SCID-PBL/hu mice. These results, demonstrate that SARS M and N DNA vaccines induced human CTL and human T-cell proliferative responses. On the other hand, we have developed SARS DNA vaccines that induce human neutralizing antibodies and human monoclonal antibodies against SARS CoV. Transgenic mice expressing SARS-CoV receptor (angiotensin converting enzyme 2) are also under development. These vaccines are expected to induce immune responses specific for SARS CoV in human and should provide useful tool for development of protective vaccines.

Impact of branching angle on neointimal coverage of drug-eluting stents implanted in bifurcation lesions.

OBJECTIVES: To investigate the impact of branching angle (BA) on neointimal coverage of drug-eluting stents (DESs) in bifurcation lesions. BACKGROUND: Previous experimental studies indicated that BA influences the local flow turbulence and wall shear stress, which are associated with neointimal coverage of DESs. METHODS: Fifty-five bifurcation lesions in 47 patients were evaluated by serial optical coherence tomography (OCT) before DES implantation and at follow-up. Neointimal coverage was assessed in cross-sectional OCT images containing the side branch; regions including the side branch ostium (SO) and vessel wall (VW) were assessed separately. BA was measured using angiography (Angio-BA) and longitudinal OCT imaging (OCT-BA). RESULTS: In the SO region, a significant negative correlation was found between the uncovered strut percentage and Angio-BA or OCT-BA (r=-0.41, P=0.0024; r=-0.33, P=0.0167, respectively) and a significant positive correlation was found between Angio-BA and average neointimal thickness (r=0.31, P=0.025), whereas no correlation was observed between OCT-BA and average neointimal thickness (r=0.20, P=0.158). In the VW region, no correlation was found between Angio-BA or OCT-BA and the uncovered strut percentage or average neointimal thickness. CONCLUSION: BA influence the neointimal coverage over DES struts in the SO at coronary bifurcation lesions, but not in those attached to the VW.

A case of midline cervical cleft.

Midline cervical cleft is a rare congenital developmental anomaly of the ventral neck. Less than 100 cases have been reported in published journals to date (Ayache et al., 1997). It is usually found as congenital scar-like skin defect or cord-like contractive abnormality of the skin at the ventral neck. Unlike "median cervical cyst" or "lateral cervical cyst" midline cervical cleft usually has no anatomical association with the hyoid bone. We will present a case of midline cervical cleft without fistula but with very small protuberant tissue. The subject was operated at the age of 5 months. We will discuss the clinical aspect and surgical management of this infrequent anomaly.

The study of novel DNA vaccines against tuberculosis: induction of pathogen-specific CTL in the mouse and monkey models of tuberculosis.

RESULTS: HSP65 + IL-12 DNA vaccine showed higher protective efficacy compared with BCG in both mouse and monkey models of TB. It induced the TB-specific CTL in the mouse model of TB, while little level of activity was observed after the injection of BCG. It also showed strong therapeutic efficacy against MDR-TB. In the monkey model, the vaccine augmented the production of IFN-γ and IL-2 from PBL and the therapeutic effect was correlated with the level of IL-2. We next evaluated the potential of DNA vaccine encoding a granulysin, which is an important defensive molecule expressed by human T cells. We found that granulysin-encoding vaccine induced the differentiation of the CTL in vitro and in vivo. It also showed therapeutic efficacy against TB in the monkey as well as the mouse model. The DNA vaccine encoding a Ksp37 also induced the TB-specific CTL in vitro and in vivo in the mouse model. It augmented the production of IL-2, IFN-γ and IL-6 from T cells and spleen cells. A synergistic effect on the activation of the TB-specific CTL was observed by the combination of Ksp37 DNA vaccine with granulysin DNA vaccine. PURPOSE AND METHODS: Emergence of the multi-drug resistant (MDR) Mycobacterium tuberculosis (TB) is a big problem in the world. We have developed novel TB vaccines [DNA vaccines encoding HSP65 + IL-12, granulysin or killer-specific secretory protein of 37kDa (Ksp37)] using Hemagglutinating virus of Japan -envelope (HVJ-E). It is suggested that the activity of the TB-specific CTL is one of the most important factor for the resistance to TB and immunity for TB in chronic human TB disease. Therefore, we examined the level of activation of the TB-specific CTL after the administration of these vaccines. CONCLUSION: These data indicate that our novel vaccines (HSP65 + IL-12 DNA, granulysin and Ksp37) have a capability to activate the TB-specific CTL and will be very strong protective and therapeutic vaccines against TB.

Novel therapeutic vaccines [(HSP65 + IL-12)DNA-, granulysin- and Ksp37-vaccine] against tuberculosis and synergistic effects in the combination with chemotherapy.

PURPOSE: Multi-drug resistant tuberculosis (MDR-TB) and extremely drug resistant (XDR) TB are big problems in the world. We have developed novel TB therapeutic vaccines, HVJ-Envelope/HSP65 + IL-12 DNA vaccine (HSP65-vaccine), granulysin vaccine and killer specific secretory protein of 37kDa (Ksp37) vaccine. METHODS AND RESULTS: HSP65 vaccine showed strong therapeutic effect against both MDR-TB and XDR-TB in mice. Intradermal immunization of HSP65-vaccine showed stronger therapeutic effect against TB than intramuscular or subcutaneous immunization. Furthermore, the synergistic therapeutic effect was observed when the vaccine was administrated in combination with Isoniazid (INH), which is a first line drug for chemotherapy. The combination of types of vaccines (HSP65- and granulysin- vaccines) also showed synergistic therapeutic effect. In the monkey model, granulysin-vaccine prolonged the survival period after the infection of TB and long-term survival was observed in vaccine-treated group. We examined the potential of two kinds of novel DNA vaccines (Ksp37-vaccine and granulysin-vaccine). Both vaccines augmented in vivo differentiation of CTL against TB. We measured the amount of Ksp37 protein in human serum and revealed that the level of Ksp37 protein of patients with tuberculosis was lower than that of healthy volunteers. Therefore, we established Ksp37 transgenic mice as well as granulysin transgenic mice to elucidate the function of those proteins. Both transgenic mice were resistant to TB infection. CONCLUSION: These data indicate the potential of combinational therapy; the combination of two DNA vaccines or combination of DNA vaccine with antibiotic drug. Thus, it will provide a novel strategy for the treatment of MDR-TB.

Novel prophylactic and therapeutic vaccine against tuberculosis.

We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-envelope and -liposome (HSP65+IL-12/HVJ). This vaccine provided therapeutic efficacy as well as remarkable protective efficacy via CD8(+) T and CD4(+) T cells in murine models compared with the saline controls, on the basis of CFU of number of multi-drug resistant TB (MDR-TB), and survival of extremely drug resistant TB (XDR-TB) challenged mice. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This vaccine exerted therapeutic efficacy (survival and immune responses) in the TB-infected monkeys. These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical trials.

Quantitative glycomics of human whole serum glycoproteins based on the standardized protocol for liberating N-glycans.

Global glycomics of human whole serum glycoproteins appears to be an innovative and comprehensive approach to identify surrogate non-invasive biomarkers for various diseases. Despite the fact that quantitative glycomics is premised on highly efficient and reproducible oligosaccharide liberation from human serum glycoproteins, it should be noted that there is no validated protocol for which deglycosylation efficiency is proven to be quantitative. To establish a standard procedure to evaluate N-glycan release from whole human serum glycoproteins by peptide-N-glycosidase F (PNGase F) treatment, we determined the efficiencies of major N-glycan liberation from serum glycoproteins in the presence of reducing agents, surfactants, protease treatment, or combinations of pretreatments prior to PNGase F digestion. We show that de-N-glycosylation efficiency differed significantly depending on the condition used, indicative of the importance of a standardized protocol for the accumulation and comparison of glycomics data. Maximal de-N-glycosylation was achieved when serum was subjected to reductive alkylation in the presence of 2-hydroxyl-3-sulfopropyl dodecanoate, a surfactant used for solubilizing proteins, or related analogues, followed by tryptic digestion prior to PNGase F treatment. An optimized de-N-glycosylation protocol permitted relative and absolute quantitation of up to 34 major N-glycans present in serum glycoproteins of normal subjects for the first time. Moreover PNGase F-catalyzed de-N-glycosylation of whole serum glycoproteins was characterized kinetically, allowing accurate simulation of PNGase F-catalyzed de-N-glycosylation required for clinical glycomics using human serum samples. The results of the current study may provide a firm basis to identify new diagnostic markers based on serum glycomics analysis.

One-pot solid-phase glycoblotting and probing by transoximization for high-throughput glycomics and glycoproteomics.

The development of rapid and efficient methods for high-throughput protein glycomics is of growing importance because the glycoform-focused reverse proteomics/genomics strategy will greatly contribute to the discovery of novel biomarkers closely related to cellular development, differentiation, growth, and aging as well as a variety of diseases such as cancers and viral infection. Recently, we communicated that rapid and efficient purification of carbohydrates can be achieved by employing sugar-specific chemical ligation with aminooxy-functionalized polymers, which we termed "glycoblotting" (see S.-I. Nishimura et al., Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 91-96). The chemoselective blotting of oligosaccharides present in crude biological materials onto synthetic polymers relies on the unique oxime-bond formation between aminooxy group displayed on the supporting materials and aldehyde/ketone group at the reducing terminal of all oligosaccharides, thus enabling highly selective and rapid oligosaccharide purification. Aiming to improve the detection sensitivity of the released oligosaccharides, we introduce here a novel strategy for one-pot solid-phase glycoblotting and probing by transoximization. We found that oligosaccharides captured by the polymer supports via the oxime bond can be released in the presence of excess O-substituted aminooxy derivatives in a weakly acidic condition. The released oligosaccharides could be recovered as newly formed oxime derivatives of the O-substituted aminooxy compound added, thus demonstrating the simultaneous releasing and probing. In addition, we synthesized a novel aminooxy-functionalized monomer, N-[2-[2-(2-tert-butoxycarbonylaminooxyacetylamino-ethoxy)ethoxy]ethyl]-2-methacrylamide, which allows for the large-scale preparation of a versatile polymer characterized by its high stability, high blotting capacity, and easy use. The one-pot protocol allowed to profile 23 kinds of N-glycan chains of human serum glycoproteins. This concept was further applied for the glycopeptides analysis in a crude mixture followed by galactose oxidase treatment to generate free aldehyde group at the non-reducing terminal of oligosaccharide moiety of glycopeptides. Our technique may be implemented in existing biochemistry and molecular diagnostics laboratories because enriched oligosaccharides and glycopeptides by solid-phase transoximization with high-sensitive labeling reagents are widely applicable in a variety of common analytical methods using two-dimensional HPLC, LC/MS, and capillary electrophoresis as well as modern mass spectrometry.

Development of vaccines and passive immunotherapy against SARS corona virus using SCID-PBL/hu mouse models.

We have investigated novel vaccine strategies against severe acute respiratory syndrome (SARS) CoV using cDNA constructs encoding the structural antigens: (S), (M), (E), or (N) protein, derived from SARS CoV. PBL from healthy human volunteers were administered i.p. into IL-2 receptor gamma-chain disrupted SCID mice, and SCID-PBL/hu mice were constructed. These mice can be used to analyze the human immune response in vivo. SARS M DNA vaccine and N DNA vaccine induced human CTL specific for SARS CoV antigens. Alternatively, SARS M DNA vaccines inducing human neutralizing antibodies and human monoclonal antibodies against SARS CoV are now being developed. These results show that these vaccines can induce virus-specific immune responses and should provide a useful tool for development of protective and therapeutic vaccines.

Evaluation of a novel vaccine (HVJ-liposome/HSP65 DNA+IL-12 DNA) against tuberculosis using the cynomolgus monkey model of TB.

We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-liposome (HSP65+IL-12/HVJ). This vaccine provided remarkable protective efficacy in mouse and guinea pig models compared to the BCG vaccine, on the basis of an induction of the CTL activity and improvement of the histopathological tuberculosis lesions, respectively. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses. Furthermore, the combination of HSP65+IL-12/HVJ and BCG by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis for human clinical trials.

DNA vaccine using hemagglutinating virus of Japan-liposome encapsulating combination encoding mycobacterial heat shock protein 65 and interleukin-12 confers protection against Mycobacterium tuberculosis by T cell activation.

We investigated the immunogenicity and protective efficacy of DNA vaccine combinations expressing mycobacterial heat shock protein 65 (Hsp65) and interleukin-12 (IL-12) using gene gun bombardment and the hemagglutinating virus of Japan (HVJ)-liposome method. A mouse IL-12 expression vector (mIL-12 DNA) encoding single-chain IL-12 proteins comprised of p40 and p35 subunits were constructed. In a mouse model, a single gene gun vaccination with the combination of Hsp65 DNA and mIL-12 DNA provided a remarkably high degree of protection against challenge with virulent Mycobacterium tuberculosis; bacterial numbers were 100-fold lower in the lungs compared to BCG-vaccinated mice. To explore the clinical use of the DNA vaccines, we evaluated HVJ-liposome encapsulated Hsp65 DNA and mIL-12DNA (Hsp65 + mIL-12/HVJ). The HVJ-liposome method improved the protective efficacy of the Hsp65 DNA vaccine compared to gene gun vaccination. Hsp65 + mIL-12/HVJ induced CD8+ cytotoxic T lymphocyte activity against Hsp65 antigen. Most importantly, Hsp65+mIL-12/HVJ vaccination resulted in a greater degree of protection than that evoked by BCG. This protective efficacy was associated with the emergence of IFN-gamma-secreting T cells and activation of proliferative T cells and cytokines (IFN-gamma and IL-2) production upon stimulation with Hsp65 and antigens from M. tuberculosis. These results suggest that Hsp65 + IL-12/HVJ could be a promising candidate for a new tuberculosis DNA vaccine, which is superior to BCG vaccine.

The development of vaccines against SARS corona virus in mice and SCID-PBL/hu mice.

We have investigated to develop novel vaccines against SARS CoV using cDNA constructs encoding the structural antigen; spike protein (S), membrane protein (M), envelope protein (E), or nucleocapsid (N) protein, derived from SARS CoV. Mice vaccinated with SARS-N or -M DNA using pcDNA 3.1(+) plasmid vector showed T cell immune responses (CTL induction and proliferation) against N or M protein, respectively. CTL responses were also detected to SARS DNA-transfected type II alveolar epithelial cells (T7 cell clone), which are thought to be initial target cells for SARS virus infection in human. To determine whether these DNA vaccines could induce T cell immune responses in humans as well as in mice, SCID-PBL/hu mice was immunized with these DNA vaccines. As expected, virus-specific CTL responses and T cell proliferation were induced from human T cells. SARS-N and SARS-M DNA vaccines and SCID-PBL/hu mouse model will be important in the development of protective vaccines.