Tuberculosis Antigen 85A Research Articles

Serum antibody profiles in individuals with latent Mycobacterium tuberculosis infection.

One‐third of the world's humans has latent tuberculosis infection (LTBI), representing a large pool of potentially active TB. Recent LTBI carries a higher risk of disease progression than remote LTBI. Recent studies suggest important roles of antibodies in TB pathology, prompting us to investigate serum antibody profiles in a cohort with LTBI. In this single‐center prospective observational study, we analyzed IgG‐antibody concentrations against five major Mycobacterium tuberculosis (Mtb) antigens (including 6 kDa early secretory antigenic target (ESAT6), CFP10, and antigen 85A, which are expressed mainly in the growth phase; and mycobacterial DNA‐binding protein 1 (MDP1) and alpha‐crystallin like protein (Acr), which are expressed in the dormant phases) in individuals with recent (n=13) or remote (n=12) LTBI, no Mtb infection (n=19), or active TB (n=15). Antibody titers against ESAT6 and MDP1 were significantly higher in individuals with recent LTBI than in those with no Mtb infection or remote LTBI. All pairwise antibody titers against these five major antigens were significantly correlated throughout the stages of Mtb infection. Five individuals with recent LTBI had significantly higher antibody titers against ESAT6 (P = 0.03), Ag85A (P = 0.048), Acr (P = 0.057), and MDP1 (P = 0.0001) than in individuals with remote LTBI; they were also outside the normal range (+2 SDs). One of these individuals was diagnosed with active pulmonary TB at 18‐month follow‐up examination. These findings indicated that concentrations of antibodies against both multiplying and dormant Mtb are higher in recent LTBI and that individuals with markedly higher antibody titers may be appropriate candidates for prophylactic therapy.

Down selecting adjuvanted vaccine formulations: a comparative method for harmonized evaluation

BackgroundThe need for rapid and accurate comparison of panels of adjuvanted vaccine formulations and subsequent rational down selection, presents several challenges for modern vaccine development. Here we describe a method which may enable vaccine and adjuvant developers to compare antigen/adjuvant combinations in a harmonized fashion. Three reference antigens: Plasmodium falciparum apical membrane antigen 1 (AMA1), hepatitis B virus surface antigen (HBsAg), and Mycobacterium tuberculosis antigen 85A (Ag85A), were selected as model antigens and were each formulated with three adjuvants: aluminium oxyhydroxide, squalene-in-water emulsion, and a liposome formulation mixed with the purified saponin fraction QS21.ResultsThe nine antigen/adjuvant formulations were assessed for stability and immunogenicity in mice in order to provide benchmarks against which other formulations could be compared, in order to assist subsequent down selection of adjuvanted vaccines. Furthermore, mouse cellular immune responses were analyzed by measuring IFN-γ and IL-5 production in splenocytes by ELISPOT, and humoral responses were determined by antigen-specific ELISA, where levels of total IgG, IgG1, IgG2b and IgG2c in serum samples were determined.ConclusionsThe reference antigens and adjuvants described in this study, which span a spectrum of immune responses, are of potential use as tools to act as points of reference in vaccine development studies. The harmonized methodology described herein may be used as a tool for adjuvant/antigen comparison studies.

Novel adjuvant for immunization against tuberculosis: DNA vaccine expressing Mycobacterium tuberculosis antigen 85A and interleukin-15 fusion product elicits strong immune responses in mice.

To investigate the potential of interleukin (IL)-15 as a novel adjuvant for Mycobacterium tuberculosis (Mtb) antigen 85A (Ag85A) vaccine. C57BL/6 mice were intramuscularly immunized three times with a plasmid expressing the Ag85A-IL-15 fusion protein (pcDNA3.1-Ag85A-IL-15), with the empty pcDNA3.1 vector and the pcDNA3.1-Ag85A as control. Mice vaccinated with pcDNA3.1-Ag85A-IL-15 generated more secretory IgA (sIgA) into their lung (209±21μg/ml) and acquired an enhanced serum IgG response to Ag85A. IgG2a/IgG1 ratios were upregulated, natural killer cell activity was augmented and Ag85A-specific splenic T cell proliferation was enhanced in these mice as well. Vaccination with pcDNA3.1-Ag85A-IL-15 promoted the polarization of CD4+ T cells towards a Th1 type in the spleen, and significantly upregulated the serum level of interferon (IFN)-γ (458±98pg/ml), a typical Th1 cytokine. IFN-γ-expressing CD8+ cells were also increased in the spleen after pcDNA3.1-Ag85A-IL-15 immunization. A superior immune type I response in mice vaccinated with plasmid Ag85A-IL-15 has been achieved.

Early Diagnosis of Tuberculosis-Associated IgA Nephropathy with ESAT-6.

IgA nephropathy (IgAN) is the most common cause of primary renal diseases worldwide, and the early secreted antigenic target of 6 (ESAT-6) which was secreted by Mycobacterium tuberculosis (MTB) may be involved in the development and progression of IgAN. This study aimed to investigate the role of ESAT-6 for early diagnosis of IgAN caused by MTB infection. From 2011 to 2014, 21 patients with renal tuberculosis (RTB), 25 with IgAN, and 46 with IgAN infected with MTB (IgAN/MTB) were enrolled. Serum levels of antibodies against Mycobacterium tuberculosis antigen 85A (Ag85A) were measured by ELISA. Urine culture and phage amplified biologically assay were performed to detect MTB. HE staining was used to observe the morphological changes in kidney tissues. Immunohistochemistry was applied to detect the expression of ESAT-6. Immunofluorescence staining was conducted to detect IgA1. Positive rates of serum anti-Ag85A antibody and urine culture for MTB were higher in the RTB and IgAN/MTB groups than those in the IgAN group. The positive rates of plaques were also higher in RTB and IgAN/MTB groups than the positive rate in the IgAN group. By contrast, the positive rate of ESAT-6 was lower in the IgAN group than that in the RTB group or the IgAN/MTB group, whereas the expression levels of IgA1 were higher in the IgAN and IgAN/MTB groups, compared with the RTB group. Our findings suggest that ESAT-6 and IgA1 may be helpful for early diagnosis of IgAN caused by MTB infection.

Ag85A DNA Vaccine Delivery by Nanoparticles: Influence of the Formulation Characteristics on Immune Responses.

The influence of DNA vaccine formulations on immune responses in combination with adjuvants was investigated with the aim to increase cell-mediated immunity against plasmid DNA (pDNA) encoding Mycobacterium tuberculosis antigen 85A. Different ratios of pDNA with cationic trimethyl chitosan (TMC) nanoparticles were characterized for their morphology and physicochemical characteristics (size, zeta potential, loading efficiency and pDNA release profile) applied in vitro for cellular uptake studies and in vivo, to determine the dose-dependent effects of pDNA on immune responses. A selected pDNA/TMC nanoparticle formulation was optimized by the incorporation of muramyl dipeptide (MDP) as an immunostimulatory agent. Cellular uptake investigations in vitro showed saturation to a maximum level upon the increase in the pDNA/TMC nanoparticle ratio, correlating with increasing Th1-related antibody responses up to a definite pDNA dose applied. Moreover, TMC nanoparticles induced clear polarization towards a Th1 response, indicated by IgG2c/IgG1 ratios above unity and enhanced numbers of antigen-specific IFN-γ producing T-cells in the spleen. Remarkably, the incorporation of MDP in TMC nanoparticles provoked a significant additional increase in T-cell-mediated responses induced by pDNA. In conclusion, pDNA-loaded TMC nanoparticles are capable of provoking strong Th1-type cellular and humoral immune responses, with the potential to be further optimized by the incorporation of MDP.

Immunization with different formulations of Mycobacterium tuberculosis antigen 85A induces immune responses with different specificity and protective efficacy

To test the relative efficacy of CD4 and CD8T cells in mediating protective immunity to Mycobacterium tuberculosis (Mtb), we compared three immunization regimes designed to induce preferentially each subset. BALB/c mice were immunized intranasally (i.n.) or parenterally with antigen 85A either in a recombinant Adenoviral vector (Ad85A), as recombinant protein (r85A) or as a set of overlapping 15mer peptides (p85A). For the first time we show that i.n. immunization with overlapping 85A synthetic peptides as well as Ad85A or r85A can provide protection against Mtb challenge. For all forms of the antigen, i.n. induces greater protection against Mtb challenge than parenteral immunization. Ad85A induces a predominantly CD8T cell response against the 85A70–78 epitope, r85A a CD4 response to 85A99–118 and p85A a balanced CD4/CD8 response to the CD4 85A99–118 and CD8 85A145–152 epitopes. Immune responses to CD4 85A99–118 and CD8 85A70–78 but not CD8 85A145–152 are protective. Although Ad85A induces a strong response to the protective CD8 85A70–78 epitope, we could not induce any response to this epitope by peptide immunization. These results show that although peptide immunization can induce protective immunity to Mtb challenge, it can also induce a response to a non-protective epitope in antigen 85A, indicating that the specificity of an immune response may be more important for protection against Mtb than its magnitude. These findings have important implications for the application of such vaccines in humans.

Mucosal and Systemic Immune Responses to Mycobacterium tuberculosis Antigen 85A following Its Co-Delivery with CpG, MPLA or LTB to the Lungs in Mice

Pulmonary vaccination is a promising route for immunization against tuberculosis because the lung is the natural site of infection with Mycobacterium tuberculosis. Yet, adjuvants with a suitable safety profile need to be found to enhance mucosal immunity to recombinant antigens. The aim of this study was to evaluate the immunogenicity, the safety and the protective efficacy of a subunit vaccine composed of the immunodominant mycolyl-transferase antigen 85A (Ag85A) and one of three powerful mucosal adjuvants: the oligodeoxynucleotide containing unmethylated cytosine-phosphate-guanine motifs (CpG), the monophosphoryl lipid A of Salmonella minnesota (MPLA) or the B subunit of heat-labile enterotoxin of Escherichia coli (LTB). BALB/c mice were vaccinated in the deep lungs. Our results showed that lung administration of these adjuvants could specifically induce different types of T cell immunity. Both CpG and MPLA induced a Th-1 type immune response with significant antigen-specific IFN-γ production by spleen mononuclear cells in vitro and a tendency of increased IFN-γ in the lungs. Moreover, MPLA triggered a Th-17 response reflected by high IL-17A levels in the spleen and lungs. By contrast, LTB promoted a Th-2 biased immune response, with a production of IL-5 but not IFN-γ by spleen mononuclear cells in vitro. CpG did not induce inflammation in the lungs while LTB and MPLA showed a transient inflammation including a neutrophil influx one day after pulmonary administration. Pulmonary vaccination with Ag85A without or with MPLA or LTB tended to decrease bacterial counts in the spleen and lungs following a virulent challenge with M. tuberculosis H37Rv. In conclusion, CpG and MPLA were found to be potential adjuvants for pulmonary vaccination against tuberculosis, providing Th-1 and Th-17 immune responses and a good safety profile.

Targeting the deep lungs, Poloxamer 407 and a CpG oligonucleotide optimize immune responses to Mycobacterium tuberculosis antigen 85A following pulmonary delivery

The current Bacille Calmette–Guérin vaccine provides variable protection against tuberculosis and new vaccination approaches are urgently needed. Pulmonary vaccination could be the best way to induce a protective immunity against Mycobacterium tuberculosis as it targets its natural site of infection. The aim of this study was to investigate the potential of Poloxamer 407 (P407) combined with a CpG oligonucleotide (CpG) to enhance immune responses to M. tuberculosis antigen 85A (Ag85A) following pulmonary delivery in BALB/c mice. An additional goal of this study was to localize the optimal delivery site of Ag85A within the lungs for generating the most intense immunity. We also investigated the capacity of P407 to prolong the residence time of the antigen within the lungs and we studied the safety of the adjuvants following pulmonary delivery. Targeting the antigen to the deep lungs produced more intense specific immune responses than targeting it to the upper airways. P407 and CpG further increased humoral immune responses and splenocyte proliferation in vitro. CpG strongly increased the Th-1 immune response with high IgG2a/IgG1 ratio, high IFN-γ and TNF-α productions by spleen mononuclear cells in vitro. P407 tended to induce a Th-2 response, as indicated by the slight decrease in IgG2a/IgG1 ratio and the slight increase in IL-5 levels. The combination of P407 and CpG produced the highest Th-1 and Th-17 responses by generating IFN-γ, TNF-α, IL-2, and IL-17A cytokines. Targeting the antigen to the deep lungs and the presence of P407 increased the residence time of the antigen within the lungs. This might explain the enhancement of immune responses induced by these factors. CpG did not induce inflammation in the lungs while P407 produced a reversible alteration of the alveolo-capillary barrier. Adding CpG to P407 did not further increase this alteration of the alveolo-capillary barrier. In conclusion, delivery of Ag85A formulated in a combination of P407 and CpG to the deep lungs induced strong immune responses, with a polyfunctional T cells phenotype.

Fusion of the Mycobacterium tuberculosis Antigen 85A to an Oligomerization Domain Enhances Its Immunogenicity in Both Mice and Non-Human Primates

To prevent important infectious diseases such as tuberculosis, malaria and HIV, vaccines inducing greater T cell responses are required. In this study, we investigated whether fusion of the M. tuberculosis antigen 85A to recently described adjuvant IMX313, a hybrid avian C4bp oligomerization domain, could increase T cell responses in pre-clinical vaccine model species. In mice, the fused antigen 85A showed consistent increases in CD4+ and CD8+ T cell responses after DNA and MVA vaccination. In rhesus macaques, higher IFN-γ responses were observed in animals vaccinated with MVA-Ag85A IMX313 after both primary and secondary immunizations. In both animal models, fusion to IMX313 induced a quantitative enhancement in the response without altering its quality: multifunctional cytokines were uniformly increased and differentiation into effector and memory T cell subsets was augmented rather than skewed. An extensive in vivo characterization suggests that IMX313 improves the initiation of immune responses as an increase in antigen 85A specific cells was observed as early as day 3 after vaccination. This report demonstrates that antigen multimerization using IMX313 is a simple and effective cross-species method to improve vaccine immunogenicity with potentially broad applicability.

Immunogenicity and Protective Efficacy of Prime-Boost Regimens with Recombinant ΔureC hly + Mycobacterium bovis BCG and Modified Vaccinia Virus Ankara Expressing M. tuberculosis Antigen 85A against Murine Tuberculosis

Immunogenicity and Protective Efficacy of Prime-Boost Regimens with Recombinant ΔureC hly ⁺ Mycobacterium bovis BCG and Modified Vaccinia Virus Ankara Expressing M. tuberculosis Antigen 85A against Murine Tuberculosis

Chemokine gene expression in lung CD8 T cells correlates with protective immunity in mice immunized intra-nasally with Adenovirus-85A

BackgroundImmunization of BALB/c mice with a recombinant adenovirus expressing Mycobacterium tuberculosis (M. tuberculosis) antigen 85A (Ad85A) protects against aerosol challenge with M. tuberculosis only when it is administered intra-nasally (i.n.). Immunization with Ad85A induces a lung-resident population of activated CD8 T cells that is antigen dependent, highly activated and mediates protection by early inhibition of M. tuberculosis growth. In order to determine why the i.n. route is so effective compared to parenteral immunization, we used microarray analysis to compare gene expression profiles of pulmonary and splenic CD8 T cells after i.n. or intra-dermal (i.d.) immunization.MethodTotal RNA from CD8 T cells was isolated from lungs or spleens of mice immunized with Ad85A by the i.n. or i.d. route. The gene profiles generated from each condition were compared. Statistically significant (p ≤ 0.05) differentially expressed genes were analyzed to determine if they mapped to particular molecular functions, biological processes or pathways using Gene Ontology and Panther DB mapping tools.ResultsCD8 T cells from lungs of i.n. immunized mice expressed a large number of chemokines chemotactic for resting and activated T cells as well as activation and survival genes. Lung lymphocytes from i.n. immunized mice also express the chemokine receptor gene Cxcr6, which is thought to aid long-term retention of antigen-responding T cells in the lungs. Expression of CXCR6 on CD8 T cells was confirmed by flow cytometry.ConclusionsOur microarray analysis represents the first ex vivo study comparing gene expression profiles of CD8 T cells isolated from distinct sites after immunization with an adenoviral vector by different routes. It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d. CD8 T cells. The sustained expression of chemokines and activation genes enables CD8 T cells to remain in the lungs for extended periods after i.n. immunization. This may account for the early inhibition of M. tuberculosis growth observed in Ad85A i.n. immunized mice and explain the effectiveness of i.n. compared to parenteral immunization with this viral vector.

Nasal associated lymphoid tissue (NALT) contributes little to protection against aerosol challenge with Mycobacterium tuberculosis after immunisation with a recombinant adenoviral vaccine

Intra-nasal administration of a recombinant adenovirus expressing Mycobacterium tuberculosis antigen 85A (Ad85A) has been shown to provide protection against challenge with M. tuberculosis. However the role of the upper respiratory tract associated lymphoid tissue, specifically the nasal associated lymphoid tissue (NALT), in providing protection has yet to be elucidated. Here we administered Ad85A to BALB/c mice alone or following BCG priming, using intranasal inocula targeting the whole respiratory tract or only the NALT, to show that Ad85A induces an immune response in the NALT insufficient to provide protection. Rather, Ad85A delivered through the respiratory tract must induce a deep lung immune response in order to protect against M. tuberculosis.

Immunogenicity and protective efficacy of mycobacterial DNA vaccines incorporating plasmid‐encoded cytokines against Mycobacterium bovis

DNA-based vaccines, alone or in combination with other sub-unit vaccination regimes, represent an alternative to live mycobacterial vaccines for protective immunization against tuberculosis. Here, we have used a murine immunization or Mycobacterium bovis aerosol challenge model to assess the immunogenicity and protective efficacy of mycobacterial DNA vaccines. Mice that received immunization with DNA constructs encoding M. bovis antigen 85A (Ag85-A) and arget(ESAT-6) produced measurable interferon-gamma (IFN-gamma) responses to CD4(+) T-cell epitope-peptide recall antigens in vitro. The magnitude of these responses was enhanced by co-delivery of a construct encoding murine cytokines (macrophage inhibitory protein (MIP)-1 alpha or interleukin(IL)-7), although they did not the match responses observed in mice that received Bacille Calmette-Guerin(BCG) immunisation. In contrast, DNA priming followed by boosting with modified vaccinia Ankara (MVA) vaccine (expressing M. tuberculosis Ag85-A) invoked higher IFN-gamma levels, with the most immunogenic regime of Ag85 or ESAT or IL-7 prime followed by MVA boost being of commensurate immunogenicity to BCG. Despite this, neither DNA alone nor DNA-prime or MVA boost regimes conferred measurable protection against aerosol challenge with virulent M. bovis. These data highlight both the promise and the shortcomings of new generation subunit tuberculosis vaccines, with particular emphasis on their potential as vaccines against M. bovis.

Investigation of immunogenic effect of the BCG priming and Ag85A- GM-CSF boosting in Balb/c mice model

Mycobacterium tuberculosis (M. tuberculosis) has once again become a major public health threat owing to the combined effects of a worldwide anti-tuberculosis drug resistance and the emergence of the human immunodeficiency virus pandemic. The Bacille Calmette-Guérin (BCG)-based vaccine has displayed inconsistent efficacy in different trials although it continues to be used to prevent tuberculosis in many countries. In current work, we have developed DNA vaccines expressing M. tuberculosis antigen 85A (Ag85A) and cytokine granulocyte macrophage colony stimulating factor (GM-CSF) and aimed to investigate the immune effect in mice based on BCG priming and DNA vaccine boosting and immune protection against M. tuberculosis challenge. Our results showed that the activity of cytotoxic T lymphocyte and spleen cell proliferative responses to Ag85A and IFN-gamma level as well as the specific antibody titer against Ag85A were significantly increased in mice immunized with prime-boost strategy in comparison with the mice immunized with BCG or DNA vaccine expressing Ag85A and GM-CSF alone. Meanwhile, the immune strategy of BCG-prime and DNA vaccine boost induced mice to generate efficient immune protection against M. tuberculosis challenge. Our data demonstrate that BCG-prime and DNA vaccine expressing Ag85A and GM-CSF boost provides a rational strategy for further development of DNA vaccine against M. tuberculosis infection.

Immunogenicity and Protective Efficacy of Prime-Boost Regimens with Recombinant Δ ureC hly + Mycobacterium bovis BCG and Modified Vaccinia Virus Ankara Expressing M. tuberculosis Antigen 85A against Murine Tuberculosis

In the light of the recent emergence of multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis, the epidemic of tuberculosis (TB) in populations coinfected with human immunodeficiency virus, and the failure of Mycobacterium bovis bacillus Calmette-Guerin (BCG) to protect against disease, new vaccines against TB are urgently needed. Two promising new vaccine candidates are the recombinant DeltaureC hly(+) BCG (recBCG), which has been developed to replace the current BCG vaccine strain, and modified vaccinia virus Ankara (MVA) expressing M. tuberculosis antigen 85A (MVA85A), which is a leading candidate vaccine designed to boost the protective efficacy of BCG. In the present study, we examined the effect of MVA85A boosting on the protection afforded at 12 weeks postchallenge by BCG and recBCG by using bacterial CFU as an efficacy readout. recBCG-immunized mice were significantly better protected against aerosol challenge with M. tuberculosis than mice immunized with the parental strain of BCG. Intradermal boosting with MVA85A did not reduce the bacterial burden any further. In order to identify a marker for the development of a protective immune response against M. tuberculosis challenge, we analyzed splenocytes after priming or prime-boosting by using intracytoplasmic cytokine staining and assays for cytokine secretion. Boosting with MVA85A, but not priming with BCG or recBCG, greatly increased the antigen 85A-specific T-cell response, suggesting that the mechanism of protection may differ from that against BCG or recBCG. We show that the numbers of systemic multifunctional cytokine-producing cells did not correlate with protection against aerosol challenge in BALB/c mice. This emphasizes the need for new biomarkers for the evaluation of TB vaccine efficacy.

Boosting BCG vaccination with MVA85A down-regulates the immunoregulatory cytokine TGF-β1

In clinical trials recombinant-modified vaccinia virus Ankara expressing the Mycobacterium tuberculosis antigen 85A (MVA85A) induces approximately 10 times more effector T cells than any other recombinant MVA vaccine. We have found that in BCG primed subjects MVA85A vaccination reduces transforming growth factor beta 1 (TGF-β1) mRNA in peripheral blood lymphocytes and reduces TGF-β1 protein in the serum, but increases IFN-γ ELISPOT responses to the recall antigen SK/SD. TGF-β1 is essential for the generation of regulatory T cells and we see a correlation across vaccinees between CD4+CD25hiFoxP3+ cells and TGF-β1 serum levels. This apparent ability to counteract regulatory T cell effects suggests a potential use of MVA85A as an adjuvant for less immunogenic vaccines.

A Simple Immunomonitoring Procedure for mRNA-Loaded Dendritic Cell Therapy

To develop a simple immunomonitoring method for dendritic cell therapy using messenger RNA (mRNA) as antigen, we evaluated whether Mycobacterium tuberculosis antigen 85A (Ag85A) mRNA-transfected peripheral blood mononuclear cells (PBMCs) could be used to stimulate the induction of interferon (IFN)-γ-producing T cells. PBMCs from 10 healthy donors were cocultured with autologous PBMCs transfected with mRNA overnight, and the number of IFN-γ-producing T cells was measured by flow cytometry. IFN-γ-producing CD4+ and CD8+ T cells were detected in 4 and 5 donors, respectively. PBMCs from 3 donors with negative results were then cocultured with Ag85A mRNA-transfected autologous PBMCs for 1 week to achieve in vitro primary induction of Ag85A-specific T cells. After restimulation with freshly prepared stimulator cells, a small but significant number of IFN-γ-producing CD8+ T cells was detected. The induction of IFN-γ-producing CD8+ T cells by overnight coculture was completely abolished by anti-class II or anti-interleukin-12 antibodies, whereas it was partially inhibited by anti-class I antibody. These data suggest that Ag85A mRNA-transfected PBMCs induce specific IFN-γ-producing T cells and might be applicable for immunomonitoring of mRNA-loaded dendritic cell therapy.

Development of Cell-Based Tuberculosis Vaccines: Genetically Modified Dendritic Cell Vaccine Is a Much More Potent Activator of CD4 and CD8 T Cells Than Peptide- or Protein-Loaded Counterparts

Genetically modified dendritic cell (DC)-based vaccines have not been explored for immunization against tuberculosis. A gene-modified DC vaccine expressing Mycobacterium tuberculosis (M.tb) antigen 85A (Ag85A) was developed by using a recombinant replication-deficient adenoviral gene transfer vector (AdAg85A). AdAg85A-transduced DC vaccine (AdAg85/DC) expressed higher levels of IL-12 and was much more immunogenic than Ag85 protein-loaded (pro/DC) or CD4/CD8 T cell peptide-loaded (pep/DC) DC vaccines. Compared to pro/DC or pep/DC, AdAg85/DC elicited a remarkably higher level of ex vivo IFN-γ production by CD4 and CD8 T cells at weeks 2, 6, and 12 postimmunization, which was coupled with higher frequencies of antigen-specific T cells. By an in vivo CD8 or CD4 T cell cytotoxicity (CTL) assay, AdAg85/DC was shown to provoke much higher and more sustained levels of CD8 and CD4 CTL activity up to 12 weeks postimmunization. Intramuscular (im) AdAg85/DC immunization was more potent than the iv route of AdAg85/DC immunization. Such stronger immunogenicity of im AdAg85/DC vaccination was corroborated with better protection from M.tb challenge. Our results thus suggest that genetically modified DC-based TB vaccine is superior to subunit DC vaccines and has the potential for therapeutic applications.