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 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.

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 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.

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.

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.

Chrysophanol glycosides from callus cultures of monocotyledonous Kniphofia spp. (Asphodelaceae).

We established callus cultures of the monocotyledonous plants Kniphofia foliosa and K. tuckii (Asphodelaceae), which produce the anthraquinone derivatives chrysophanol and its glycosides. The minor product chrysophanol 8-O-beta-gentiobioside was fully characterized by spectroscopic analysis and synthesis.