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.

Results: HSP 65 + 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-gamma. 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-gamma 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 HSP 65 + 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 (HSP 65 + 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.

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.

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.

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 (4 month) 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 15 kD (15 K) granulysin protein and mRNA in CD8 positive T cells in the patients infected with drug sensitive (TB) or multidrug resistant M. tuberculosis (MDR-TB) 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 (15 K granulysin transgenic mice and 9 K granulysin transgenic mice). It was demonstrated that 15 K granulysin transgenic mice as well as 9 K 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 15 K granulysin and 9 K granulysin against TB infection. Moreover, DNA vaccine expressing 15 K 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.

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.

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). An IL-12 expression vector (IL-12DNA) encoding single-chain IL-12 proteins comprised of p40 and p35 subunits were constructed. This vaccine provided remarkable protective efficacy in mouse and guinea pig models 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 multi-drug resistant TB (MDR-TB) and extremely drug resistant TB (XDR-TB) (prolongation of survival time and the decrease in the number of TB in the lung) 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. All monkeys in the control group (saline) died within 8 months, while 50% of monkeys in the HSP65+hIL-12/HVJ group survived more than 14 months post-infection (the termination period of the experiment). 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). In contrast, 33% of monkeys from BCG Tokyo alone group were alive (33% 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.

We have developed novel therapeutic tuberculosis (TB) vaccines: IL-6 related genes (IL-6 gene + IL-6 receptor gene + gp130 gene using adenovirus vector) and granulysin DNA gene. IL-6 related DNA vaccine provided remarkable therapeutic efficacy in BALB/c mouse, on the basis of an induction of the CTL activity and improvement of the histopathological tuberculosis lesions, respectively. The number of live TB in lungs from mice treated with IL-6 related genes therapy was smaller than that from the control mice treated with BCG Tokyo. CTL activity against TB was enhanced by the assay for detecting the production of IFN-g from effecter lymphocytes stimulated with PPD or killed TB. The histopathological findings in the lungs derived from mice treated with IL-6 related genes were improved. Furthermore, we established granulysin DNA vaccine. Human granulysin cDNA was constructed into pcDLSRα DNA. The number of live TB in lungs, spleens and liver treated with granulysin DNA therapy even after the challenge of TB was smaller than that from the control mice (non-treated mice). Synergistic therapeutic effect of granulysin DNA and IL-6 realated genes is now being studied. These vaccines should provide strong tools for therapy against Multi-drug resistant tuberculosis. [H-17-Shinko-5] of Research on Emerging and Re-emerging Infectious Diseases in Health Sciences Research grants from Japan.

LB-307 In previous study we showed synergistic effect of IL-6 related genes (IL-6 gene, IL-6 receptor gene and gp130 gene) on the induction of CTL against human cancer cells using CB17-SCID-PBL/hu experimental model. In this model IL-6 related genes were administered in vivo using adenovirus vector into CB17-SCID-PBL/hu mice bearing human cancers, and induced CTL activity against human tumor cells (lung cancer, stomach cancer, colon cancer) and significant prolonged survival of mice was observed. IL-2 receptor γ-chain disrupted NOD-SCID mice have little activity of NK cell. Therefore, the engraftment of human T cell in IL-2 receptor γ-chain disrupted NOD-SCID [IL-2R (-/-) SCID] is very stronger than that in CB17-SCID mice. IL-6 related genes were injected (i-p) into IL-2R (-/-) SCID-PBL/hu mice in vivo by using this experimental model [IL-2R(-/-)SCID mice given PBL from healthy volunteer and CESS human cancer cells]. Forty percent of spleen cells in these IL-2R (-/-) SCID-PBL/hu mice were CD3 + human T cell. Two weeks after the immunization with CESS cells, 50% of cytotoxic activity against CESS cells was detected in spleen cells at an E/T ratio of 10:1. The CTL (CD4 - CD8 + CD3 +) human T activity in the spleen cells from IL-2R(-/-) SCID-PBL/hu was extremely higher (more than 100 fold) than that from CB-17 SCID-PBL/hu. Furthermore, recombinant granulysin exerted strong cytotoxic activity in vitro against human lung cancer cell lines. To our knowledge, it is quite difficult to generate the invivo CTL against tumor associate antigens in human treated with several kinds of cancer vaccines (tumor associated antigens vaccines). Therefore, the recombinant granulysin and the IL-2R(-/-) SCID-PBL/hu model using gene encoding lung cancer associated antigens or peptides of tumor associated antigens might provide a powerful weapon for developing new cancer vaccines against human lung cancers. (Co-worker: Izumu Saito: Tokyo University) (Grant sponsor: Ministry of Health Labor and Welfare, Japan)

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 γ-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.

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.

Abstract 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 BALB/C 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, respetively. This vaccine strongly induced CTL against HSP antigen and M.tuberculosis antigens 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 and immune responses. 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). In contrast, the survival of the BCG Tokyo group was 33%. These data indicate that our novel DNA vaccine might be useful against M.tuberculosis for human clinical trials. [H-17-Shinko-5] of Research on Emerging and Re-emerging Infectious Diseases in Health Sciences Research grants from Japan.