Mtb32 is a promising tuberculosis antigen for DNA vaccination in pre- and post-exposure mouse models.

Identification of antigens that provide protective immunity via prophylactic and therapeutic vaccination against Mycobacterium tuberculosis is critical for the development of subunit vaccines for tuberculosis (TB). In this study, we performed a head-to-head comparison of seven well-known TB antigens delivered by DNA vaccine, and evaluated their respective immunogenicities and protective efficacies in pre- and post-exposure mouse models. All TB antigens were designed as a chimeric fusion with Flt3-L to enhance antigen-specific T-cell immunity upon vaccination. Prophylactic vaccination with the Flt3L (F)-Mtb32 DNA vaccine elicited significant protection in both the spleen and lungs against M. tuberculosis challenge, comparable to the Bacillus Calmette-Guerin vaccine. F-Ag85A and F-Mtb32 DNA vaccines, in combination with chemotherapy, reduced the bacterial burden to undetectable levels in the lungs of all mice infected with M. tuberculosis. These data collectively indicate that the F-Mtb32 DNA vaccine confers the most efficient protective immunity that suppresses bacterial growth in the active or latent status of M. tuberculosis.

The effects of mesenchymal stem cells injected via different routes on modified IL-12-mediated antitumor activity.

Owing to its tumor tropism and prolonged transgene expression, mesenchymal stem cell (MSC) has been considered as an ideal delivery vehicle for cancer gene therapies or therapeutic vaccines. In this study, we demonstrated that intratumoral (i.t.) injection of MSCs expressing modified interleukin-12 (MSCs/IL-12M) exhibited stronger tumor-specific T-cell responses and antitumor effects as well as more sustained expressions of IL-12 and interferon (IFN)-γ in both sera and tumor sites than did IL-12M-expressing adenovirus (rAd/IL-12M) in mice bearing both solid and metastatic tumors. Subcutaneous (s.c.) injection of MSCs/IL-12M at contralateral site of tumor exhibited similar levels of serum IL-12 and IFN-γ as i.t. injection, but much weaker antitumor effects in both B16F10 melanoma and TC-1 cervical cancer models than i.t. injection. Although intravenous (i.v.) injection elicited earlier peak serum levels of cytokines, it induced weaker tumor-specific T-cell responses and antitumor effects than i.t. injection, indicating that serum cytokine levels are not surrogate indicators of antitumor effects. Taken together, these results indicated that MSC is more efficient than adenovirus as a cytokine gene delivery vehicle and that i.t. injection of MSCs/IL-12M is the best approach to induce strong tumor-specific T-cell responses that correlate with anti-metastatic effects as well as inhibition of solid tumor growth, although MSCs themselves have an ability to migrate into the tumor site. In addition, MSCs/IL-12M embedded in Matrigel (MSCs/IL-12M/Matrigel) exhibited significant antitumor effects even in immunodeficient mice such as SCID and BNX mice lacking T, B and natural killer (NK) cells, but not in IFN-γ knockout mice. Our findings provide an optimal approach for designing an efficient clinical protocol of MSC-based cytokine gene therapy to induce strong tumor-specific T-cell responses and therapeutic anticancer efficacy.

Raising medical student awareness of compassionate care through reflection of annotated videotapes of clinical encounters.

CONTEXT: With medical training focused on medical knowledge and skills, the nurturing of humanistic care can suffer. OBJECTIVES: We designed and conducted an outpatient rheumatology patient-partner exercise that integrates the assessment of student compassionate care into an outpatient clinical skills training exercise. METHODS: Eleven third-year medical students were videotaped performing a medical history on a patient volunteer. Students, the preceptor and a fourth-year medical student independently observed the videotape, tagged segments demonstrating observed or missed compassionate care opportunities and completed a compassionate care questionnaire. Students also participated in a focus group. Ten patients completed a comparable questionnaire and provided feedback on student encounters. FINDINGS: Students recognized and reflected on opportunities for compassionate care. The preceptor's feedback was reinforced. Students' ratings of their demonstrations of compassionate care were lower after reviewing videotapes, and were also lower than preceptor ratings. Patients were satisfied with the exercise and highlighted student professionalism. CONCLUSIONS: The exercise proved to be an effective format for promoting student reflection on and self-assessment of compassionate care. It demonstrated that nurturing compassionate care can be integrated into an outpatient clinical skills exercise.

Branched oligomerization of cell-permeable peptides markedly enhances the transduction efficiency of adenovirus into mesenchymal stem cells.

Cell-permeable peptides (CPPs) promote the transduction of nonpermissive cells by recombinant adenovirus (rAd) to improve the therapeutic efficacy of rAd. In this study, branched oligomerization of CPPs significantly enhanced the transduction of human mesenchymal stem cells (MSCs) by rAd in a CPP type-independent manner. In particular, tetrameric CPPs increased transduction efficiency at 3000-5000-fold lower concentrations than did monomeric CPPs. Although branched oligomerization of CPPs also increases cytotoxicity, optimal concentrations of tetrameric CPPs required for maximum transduction are at least 300-1000-fold lower than those causing 50% cytotoxicity. Furthermore, although only approximately 60% of MSCs were maximally transduced at 500 muM of monomeric CPPs, >95% of MSCs were transduced with 0.1 muM of tetrameric CPPs. Tetrameric CPPs also significantly increased the formation and net surface charge of CPP/rAd complexes, as well as the binding of rAd to cell membranes at a greater degree than did monomeric CPPs, followed by rapid internalization into MSCs. In a critical-size calvarial defect model, the inclusion of tetrameric CPPs in ex vivo transduction of rAd expressing bone morphogenetic protein 2 into MSCs promoted highly mineralized bone formation. In addition, MSCs that were transduced with rAd expressing brain-derived neurotrophic factor in the presence of tetrameric CPPs improved functional recovery in a spinal cord injury model. These results demonstrated the potential for tetrameric CPPs to provide an innovative tool for MSC-based gene therapy and for in vitro gene delivery to MSCs.

A panel of antibodies to determine site of origin and malignancy in smooth muscle tumors.

Leiomyosarcomas are malignant smooth muscle tumors that occur most commonly in the gynecologic tract and soft tissue. There are different diagnostic criteria of malignancy for smooth muscle tumors arising at gynecologic and soft tissue sites and they may be managed differently but determining the primary site of a smooth muscle tumor can be difficult in some cases. In addition, the distinction between malignant and benign gynecologic tract smooth muscle tumors on morphologic grounds can be challenging. Using a series of tissue microarrays that contain 245 cases of leiomyosarcomas (102 gynecologic) with survival data, and 49 cases of uterine leiomyoma, we examined the ability of selected immune-markers (estrogen receptor (ER) and WT1) to distinguish between leiomyosarcomas of gynecologic and nongynecologic origin. In addition, we examined whether immunostains for p16, p53 and Ki-67 could distinguish between malignant and benign gynecologic smooth muscle tumors. ER nuclear positivity was observed in 3 and 50% of the nongynecologic and gynecologic leiomyosarcomas, respectively (P<0.001). Nuclear WT1 positivity was seen in 0 and 8% of the nongynecologic and gynecologic leiomyosarcomas, respectively (P<0.001). 87% of primary gynecologic leiomyosarcomas and 2% of uterine leiomyomas showed diffuse (>or=50% of cells) p16 staining (P<0.001). 23% of gynecologic leiomyosarcomas showed p53 immunopositivity (>or=50% of cells) whereas none of the leiomyomas were positive for p53 (P<0.001). 65% of the gynecologic leiomyosarcomas and 0% of the leiomyomas exhibited >10% Ki-67 proliferation index (P<0.001). Diffuse p16 and p53 immunopositivity and high Ki-67 proliferation index, singly or in combination, yielded an overall sensitivity of 92% and specificity of 98% for distinguishing between gynecologic leiomyosarcomas and leiomyomas and can be used as indicators of malignancy for gynecologic smooth muscle tumors. Although ER positivity can be used to support the gynecologic origin of a leiomyosarcomas, nuclear WT1 immunostaining is of little use.

The cyanase operon and cyanate metabolism.

Cyanase is an inducible enzyme in E. coli that catalyzes bicarbonate-dependent decomposition of cyanate. It is encoded as part of an operon we have named the cyn operon, which includes three genes in the following order: cynT (cyanate permease), cynS (cyanase), and cynX (protein of unknown function). The direction of transcription is opposite to that of the lac operon, and the 3'-end of the cyn operon overlaps the 3'-end of the lac operon by 98 nucleotides. The gene cynR (regulatory protein) is located upstream from the cyn operon, and its transcription is opposite that of the cyn operon. The genes of the cyn operon and the cynR gene have been cloned, sequenced and over-expressed. Cyanate at concentrations of about 1 mM is toxic to strains of E. coli lacking the cyanase gene, but strains in which the inducible gene for cyanase is present can grow on cyanate as the sole source of nitrogen at concentrations as high as 20 mM. The presence of cyanase itself is not sufficient to overcome cyanate toxicity--the permease must also be present. Strains lacking the cyanase gene, but having a functional permease gene, are extremely sensitive to cyanate. Uptake of cyanate involves the product of the permease gene in an energy-dependent process. It appears that the cyn operon has evolved to function in detoxification/decomposition of cyanate arising from both intra- and extracellular sources.

NAMPT suppresses glucose deprivation-induced oxidative stress by increasing NADPH levels in breast cancer.

Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme involved in NAD+ biosynthesis. Although NAMPT has emerged as a critical regulator of metabolic stress, the underlying mechanisms by which it regulates metabolic stress in cancer cells have not been completely elucidated. In this study, we determined that breast cancer cells expressing a high level of NAMPT were resistant to cell death induced by glucose depletion. Furthermore, NAMPT inhibition suppressed tumor growth in vivo in a xenograft model. Under glucose deprivation conditions, NAMPT inhibition was found to increase the mitochondrial reactive oxygen species (ROS) level, leading to cell death. This cell death was rescued by treatment with antioxidants or NAD+. Finally, we showed that NAMPT increased the pool of NAD+ that could be converted to NADPH through the pentose phosphate pathway and inhibited the depletion of reduced glutathione under glucose deprivation. Collectively, our results suggest a novel mechanism by which tumor cells protect themselves against glucose deprivation-induced oxidative stress by utilizing NAMPT to maintain NADPH levels.

A novel Bcl-2 related gene, Bfl-1, is overexpressed in stomach cancer and preferentially expressed in bone marrow.

Programmed cell death (apoptosis) is an active process which is genetically encoded and plays an important role in several cellular activities such as embryonic development, deletion of autoreactive T-cells and homeostasis. Several genes regulating apoptosis have been reported, including p53, one of the tumor suppressor genes, c-myc, one of the proto-oncogenes, and various kinds of Bcl-2 related genes. A new cDNA clone which is homologous to Bcl-2, named as Bfl-1 were isolated from a human fetal liver at 22 week of gestation. This clone was identified by computer analysis of random cDNA sequences that were obtained in an effort to expand the expressed sequence tag (EST) databases to be used for human genome analysis. The homology was recognized by 72% amino acid identity to the murine A1 gene, a member of the Bcl-2-related genes. The homology to the BH1 and BH2 domains of Bcl-2 was especially significant, suggesting that Bfl-1 is a new member of the Bcl-2-related genes. Bfl-1 is abundantly expressed in the bone marrow and at a low level in some other tissues. Interestingly, a correlation was noted between the expression level of Bfl-1 gene and the development of stomach cancer in eight sets of clinical samples. It is conceivable that Bfl-1 is involved in the promotion of the cell survival in the stomach cancer development or progression.

HCV core protein modulates Rb pathway through pRb down-regulation and E2F-1 up-regulation.

It has been recognized that the HCV (hepatitis C virus) core protein plays an important role in hepatocarcinogenesis. The functional inactivation of the Rb pathway appears to be a major event for multi-step cancer carcinogenesis. To elucidate the role of the HCV core protein in hepatocarcinogenesis, we investigated the effect of the HCV core protein on the Rb pathway in both Rat-1 cell lines, stably expressing the HCV core protein and the doxycycline-regulated cell lines. The HCV core stable transfectants showed a dramatic decrease in the pRb levels and E2F-1 up-regulation. In the doxycycline-regulated cell lines, the pRb levels were significantly decreased which are followed by E2F-1 up-regulation. HCV core stable transfectants showed higher cell growth rates and were sensitize to apoptosis. Thus, our results first indicate that the HCV core protein decreases the expression of pRb, thereby allowing E2F-1 to be constitutively active, which is thought to result in rapid cell proliferation or sensitizing to apoptosis.

Short peptides with induced beta-turn inhibit the interaction between HIV-1 gp120 and CD4.

To identify novel peptides that inhibit the interaction between human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 and CD4, we constructed a targeted phage-displayed peptide library in which phenylalanine and proline were fixed at the fourth and sixth positions, respectively, because Phe43 and the adjacent beta-turn of CD4 are critical for interaction with gp120. Two synthetic peptides were selected after three rounds of biopanning against gp120, and one of them, G1 peptide (ARQPSFDLQCGF), exhibited specific inhibition of the interaction between gp120 and CD4 with an IC(50) of about 50 microM. Structural analysis using NMR demonstrated that G1 peptide forms a compact cyclic structure similar to the CD4 region interacting with gp120. Two derivatives of G1 peptide, a linear hexameric peptide (G1-6) and a cyclic nonameric peptide (G1-c), were synthesized based on the structure of the G1 peptide. Interestingly, they showed higher inhibitory activities than did G1 peptide with IC(50)'s of 6 and 1 microM, respectively. Thus, this study might provide a new insight into the development of anti-HIV-1 inhibitors.

A neuron-specific gene transfer by a recombinant defective Sindbis virus.

We examined the possibility that Sindbis virus, an alpha virus with a single-stranded RNA genome, would be applied for neuronal gene transfer. The recombinant defective Sindbis viruses were constructed by replacing the structural genes of Sindbis virus with genes encoding beta-galactosidase (rdSind-lacZ) or enhanced green fluorescent protein (rdSind-EGFP). In neuron-glia cocultures prepared from the neocortex, hippocampus, and striatum, EGFP or beta-galactosidase was expressed selectively in neurons 24 h after infection with rdSind-EGFP or rdSind-lacZ. Most cortical neurons were infected with rdSind-lacZ at a multiplicity of infection (M.O.I.) of 5 while glial cells were little infected. In addition, transient neuron-specific expression of beta-galactosidase was observed near injection sites over the next 3 d following administration of rdSind-lacZ in adult rat. In the cortical neurons infected with rdSind-EGFP, treatment with NMDA induced neuritic blebs and cell body swelling in a Na+-dependent manner. Therefore, recombinant defective Sindbis viruses can be used as an efficient and selective vector for gene transfer into neurons and applied to investigate biological role of target genes delivered into neurons in vitro and in vivo.

Hepatitis C virus envelope DNA-based immunization elicits humoral and cellular immune responses.

The vaccine development for hepatitis C virus (HCV) is highly urgent to prevent non A and non B hepatitis. It was recently shown that the HCV envelope proteins appeared to the key viral antigens to induce protective immunity. To generate immune responses to the HCV envelope proteins on the DNA-based immunization, various envelope gene-containing plasmids were constructed. For efficient expression and secretion of envelope proteins, the signal sequence of each envelope protein was replaced with either herpes simplex virus type-1 (HSV-1) gD or signal sequence of gD and truncated C-terminal hydrophobic regions of envelope proteins. The intramuscular injection of these plasmids generated a significant level of antibody titers to the E1 and E2 proteins, which maximally reached 850 and 25,000 respectively. The secreted form of each envelope protein and the fusion of the highly immunogenic gD proteins were shown to have no significant effect on generating immune responses to the envelope proteins. In addition, immunized rats appeared to generate antibodies directed to the homologous HVR-1 peptide. Splenic lymphocytes from immunized rats were shown to induce significant T-cell proliferative responses with the stimulation of recombinant E1 and E2 proteins. Our results demonstrated that the HCV envelope-DNA based immunization could elicit both humoral and cellular immune responses.

Comparison of various expression plasmids for the induction of immune response by DNA immunization.

Intramuscular injection of plasmid DNA is an efficient method to introduce a foreign gene into a live animal. We investigated several factors affecting the gene transfer efficiency and the following immune response by intramuscular injection of plasmid DNA. When the strength of several highly efficient viral promoters was compared in muscle by using the chloramphenicol acetyltransferase (CAT) gene as an indicator, cytomegalovirus (CMV) immediate early promoter was found to be stronger than any other viral promoters including Rous sarcoma virus (RSV), murine leukemia virus (SL3-3) and simian virus 40 (SV40) early promoters. Inclusion of adenovirus tripartite leader (TPL) sequences and a synthetic intron in the 5' untranslated region of mRNA moderately stimulated the CAT expression. On the other hand, the expression of encephalomyocarditis virus (EMCV) VP1 gene was greatly enhanced by the TPL sequences and an intron. The level of humoral immune response by intramuscular injection of various VP1 expression plasmids was compared. The seroconversion rate was highly dependent on the strength of the expression vector. However, the ratio of IgG1 and IgG2a immune response was not significantly variable depending on the strength of the expression vector. Also, the efficiency of the sindbis virus-based DNA vector was examined for the gene expression and immune response. Although a high level of CAT expression was obtained in muscle by using this system, VP1 was not produced as much as the conventional expression vectors. Furthermore, little humoral immune response was elicited by intramuscular injection of VP1-expressing sindbis vector, suggesting that this system was not superior to the conventional vector for DNA immunization.

Induction of apoptosis in p53-deficient human hepatoma cell line by wild-type p53 gene transduction: inhibition by antioxidant.

We investigated the role of wild-type (wt)-p53 as an inducer of apoptotic cell death in human hepatoma cell lines. Following the retrovirus-mediated transduction of the wt-p53 gene, Hep3B cells lacking the endogenous p53 expression began to die through apoptosis in 4 h. They showed a maximal apoptotic death at 12 h, whereas HepG2 cells expressing endogenous p53 did not. However, the transduction of the wt-p53 gene elicited growth suppression of both Hep3B and HepG2 cells. P21(WAF1/CIP1), a p53-inducible cell cycle inhibitor, was induced, not only in Hep3B cells undergoing apoptosis, but also in HepG2 cells. The kinetics of the p21(WAF1/CIP1) induction, DNA fragmentation, and growth suppression of the Hep3B cells showed that DNA fragmentation and growth suppression progressed rapidly following p21(WAF1/CIP1) accumulation. N-acetyl-cysteine or glutathione, potent antioxidants, strongly inhibited the DNA fragmentation, but did not reduce the elevated level of p21(WAF1/CIP1). These findings suggested that p21(WAF1/CIP1) was not a critical mediator for the execution of p53-mediated apoptosis, although it contributed to the growth inhibition of cells undergoing apoptosis. Furthermore, p53-mediated apoptosis could be repressed by antioxidants.

Construction of hepatitis C-SIN virus recombinants with replicative dependency on hepatitis C virus serine protease activity.

An in vivo assay system was developed for the serine protease of hepatitis C virus (HCV) using the sindbis (SIN) viral replication system in which HCV serine protease activity is essential for the replication of the HCV-SIN chimeric virus. Two chimeric viral cDNA clones were constructed by inserting the NS3/4A region and NS3/4A region with the putative helicase deleted, into the N-terminal region of SIN core protein. The constructs were named Tpro CT and Tpro T, respectively. BHK-21 cells transfected with the in vitro transcribed RNAs from Tpro CT and Tpro T showed specific cytopathic morphology and produced chimeric viruses, Vpro CT and Vpro T. In contrast, in vitro transcribed RNAs from Tpro CTI and Tpro TI, in which serine of catalytic triad of HCV protease was changed to alanine, were not infectious. When the chimeric viruses were passaged in BHK-21 cells at about 0.1 multiplicity of infection (MOI), Vpro T, but not Vpro CT, stably expressed HCV protease for up to five passages. Surprisingly, the cell culture media of BHK-21 cells infected with Vpro T, compared to wild-type sindbis virus, showed rapid pH changes by more than 0.8 pH degree at 72 h post-infection. HCV-SIN hybrid viruses could be used in screening the HCV protease-inhibitor in cell culture systems.

In vivo assay for hepatitis C viral serine protease activity using a secreted protein.

Hepatitis C virus (HCV) is a major pathogen of community-acquired and post-transfusional non-A, non-B hepatitis. Since an in vitro replication system is not available, it is crucial to develop an efficient and sensitive assay system for screening inhibitors of HCV. The fact that the activity of HCV NS3 protease is responsible for the maturation of the nonstructural proteins and viral replication, suggests that NS3 protease is a suitable target for anti-HCV drug development. To devise an assay system in cell culture, we constructed NS3/4A-SEAP (secreted alkaline phosphatase) chimeric gene, in which the SEAP gene was fused in-frame to downstream of NS4A/4B cleavage site. In this system, the SEAP would be secreted into the extracellular media depending on the cleavage activity of the NS3 protease. Our results demonstrate that the NS3/4A-SEAP expression vector encoding wild type NS3 protease, but not mutant NS3 protease, could produce high SEAP activity in the media of both transfected cells and stable expression cell lines. Since the activity of SEAP in the culture media can be monitored quantitatively and continuously by the chemiluminescent method, this assay system will be useful for screening potential inhibitors of HCV protease.

Enhanced delivery efficiency of recombinant adenovirus into tumor and mesenchymal stem cells by a novel PTD.

Protein transduction domains (PTDs) are small peptides that facilitate the transduction of large molecules such as polyproteins, DNA and viruses into a eukaryotic cell. Here, we demonstrated that a novel PTD (HP4) derived from herring protamine appeared to enter C6Bu1 rat glioma cell lines more rapidly than other known PTDs such as Tat, Antp and Hph-1. Moreover, HP4 significantly enhanced in vitro transduction of recombinant adenoviruses (rAds) into various cancer cell lines, mesenchymal stem cells (MSCs) and dendritic cells, which are relatively resistant to rAd infection. Enhancement of rAd delivery into C6Bu1 and MSCs by HP4 is 20 and 7 times higher than that by Tat, respectively. The increase in the expression of rAd encoding IL-12N220L by HP4 is proportional to its antitumor effect in the ex vivo transduced mouse colon cancer model. Thus, these results suggest that HP4 could be utilized to improve the transduction efficiency of rAd, resulting in enhanced efficacy of rAd-mediated gene therapy, especially for ex vivo-transduced cell therapy.

Adenovirus-mediated gene transfer of interleukin-23 shows prophylactic but not therapeutic antitumor effects.

A novel cytokine interleukin (IL)-23 bears a structural and functional resemblance to IL-12. A recombinant adenovirus expressing IL-23N220L (recombinant replication-defective adenovirus (rAd)/IL-23N220L) that selectively secrets IL-23 was constructed and compared with rAd/IL-12N220L in terms of immunological and antitumor effects. In a prophylactic setting, vaccination with rAd/ovalbumin (OVA) and rAd/IL-23N220L enhanced OVA-specific CD8(+) T-cell responses that were closely associated with complete protection against the subsequent challenge of OVA-expressing E.G7 thymoma. However, in a therapeutic setting, the intratumoral injection of rAd/IL-23N220L showed only marginal antitumor activity against several established tumors such as E.G7, CT26 and B16F10. Interestingly, whereas IL-23 still induced tumor-specific CD8(+) T-cell responses, it could not activate natural killer (NK) cells in vitro and in vivo. In addition, the adoptive transfer of activated NK cells partially restored the therapeutic antitumor effect of IL-23, indicating that NK cells are one of the crucial factors responsible for the regression of established tumors. Taken together, we demonstrated that adenovirus-mediated gene transfer of IL-23 induces a potent prophylactic, but not a therapeutic, antitumor effect.

Correlation of antiviral T-cell responses with suppression of viral rebound in chronic hepatitis B carriers: a proof-of-concept study.

Despite recent advances in the chemotherapy of chronic hepatitis B (CHB), an effective viral suppression after cessation of therapy has not yet been achieved. To investigate whether hepatitis B virus (HBV)-specific T-cell responses are inducible and can contribute to the viral suppression after cessation of the therapy, we conducted a proof-of-concept study with a DNA vaccine comprising of most HBV genes plus genetically engineered interleukin-12 DNA (IL-12N222L) in 12 CHB carriers being treated with lamivudine (LAM). When the ex vivo and/or cultured IFN-gamma enzyme-linked immunospot (ELISPOT) assay was performed, the detectable HBV-specific IFN-gamma secreting T-cell responses were observed at the end of treatment and during a follow-up. These type 1T-cell responses, particularly CD4(+) memory T-cell responses could be maintained for at least 40 weeks after the therapy and correlated with virological responses, but not with alanine aminotransferase elevation. Moreover, DNA vaccination under LAM treatment appeared to be well-tolerated and showed 50% of virological response rate in CHB carriers. Thus, a combination therapy of the DNA vaccine with chemotherapy may be one of new immunotherapeutic methods for the cure of CHB.

Protective effect of DNA vaccine during chemotherapy on reactivation and reinfection of Mycobacterium tuberculosis.

Active disease of tuberculosis (TB) can be developed decades later by either a relapse of the initial infection (endogenous reactivation) or by an entrance of the secondary infection (exogenous reinfection), since the current chemotherapy cannot lead to complete elimination of tuberculosis. Although the immunotherapeutic approaches in conjunction with conventional chemotherapy were tried to prevent TB growth via boosting the immune system, their therapeutic effects are still controversial. Here, we found that TB DNA vaccination completely blocked tuberculosis reactivation and significantly prevented from the secondary infection when chemotherapy was combined simultaneously. In particular, double-gene DNA vaccine composed of Ag85A and PstS-3 genes could reduce bacteria growth better than single-gene DNA vaccine after a secondary reinfection, indicating a correlation between the breadth of Th1 IFN-gamma response and the efficacy of the protection from reinfection. Thus, we propose that multigene TB DNA immunotherapy including Ag85A and PstS-3 genes during the period of chemotherapy could benefit patients undergoing TB chemotherapy in prevention from exogenous reinfection as well as endogenous reactivation.