PO and ID BCG vaccination in humans induce distinct mucosal and systemic immune responses and CD4+ T cell transcriptomal molecular signatures.

Protective efficacy of Bacillus Calmette-Guérin (BCG) may be affected by the methods and routes of vaccine administration. We have studied the safety and immunogenicity of oral (PO) and/or intradermal (ID) administration of BCG in healthy human subjects. No major safety concerns were detected in the 68 healthy adults vaccinated with PO and/or ID BCG. Although both PO and ID BCG could induce systemic Th1 responses capable of IFN-γ production, ID BCG more strongly induced systemic Th1 responses. In contrast, stronger mucosal responses (TB-specific secretory IgA and bronchoalveolar lavage T cells) were induced by PO BCG vaccination. To generate preliminary data comparing the early gene signatures induced by mucosal and systemic BCG vaccination, CD4+ memory T cells were isolated from subsets of BCG vaccinated subjects pre- (Day 0) and post-vaccination (Days 7 and 56), rested or stimulated with BCG infected dendritic cells, and then studied by Illumina BeadArray transcriptomal analysis. Notably, distinct gene expression profiles were identified both on Day 7 and Day 56 comparing the PO and ID BCG vaccinated groups by GSEA analysis. Future correlation analyses between specific gene expression patterns and distinct mucosal and systemic immune responses induced will be highly informative for TB vaccine development.

Intranasal vaccinations with the trans-sialidase antigen plus CpG Adjuvant induce mucosal immunity protective against conjunctival Trypanosoma cruzi challenges.

Trypanosoma cruzi is an intracellular protozoan parasite capable of infecting through mucosal surfaces. Our laboratory has previously elucidated the anatomical routes of infection after both conjunctival and gastric challenge in mice. We have shown that chronically infected mice develop strong immune responses capable of protecting against subsequent rechallenge with virulent parasites through gastric, conjunctival, and systemic routes of infection. We have also shown that intranasal immunizations with the unique T. cruzi trans-sialidase (TS) antigen protect against gastric and systemic T. cruzi challenge. In the current work we have investigated the ability of purified TS adjuvanted with CpG-containing oligonucleotides to induce immunity against conjunctival T. cruzi challenge. We confirm that intranasal vaccinations with TS plus CpG induce TS-specific T-cell and secretory IgA responses. TS-specific secretory IgA was detectable in the tears of vaccinated mice, the initial body fluid that contacts the parasite during infectious conjunctival exposures. We further show that intranasal vaccinations with TS plus CpG protect against conjunctival T. cruzi challenge, limiting local parasite replication at the site of mucosal invasion and systemic parasite dissemination. We also provide the first direct evidence that mucosal antibodies induced by intranasal TS vaccination can inhibit parasite invasion.

Anatomical route of invasion and protective mucosal immunity in Trypanosoma cruzi conjunctival infection.

Trypanosoma cruzi is a protozoan parasite that can initiate mucosal infection after conjunctival exposure. The anatomical route of T. cruzi invasion and spread after conjunctival parasite contamination remains poorly characterized. In the present work we have identified the sites of initial invasion and replication after contaminative conjunctival challenges with T. cruzi metacyclic trypomastigotes using a combination of immunohistochemical and real-time PCR confirmatory techniques in 56 mice between 3 and 14 days after challenge. Our results demonstrate that the predominant route of infection involves drainage of parasites through the nasolacrimal duct into the nasal cavity. Initial parasite invasion occurs within the ductal and respiratory epithelia. After successive waves of intracellular replication and cell-to-cell spread, parasites drain via local lymphatic channels to lymph nodes and then disseminate through the blood to distant tissues. This model of conjunctival challenge was used to identify immune responses associated with protection against mucosal infection. Preceding mucosal infection induces mucosal immunity, resulting in at least 50-fold reductions in recoverable tissue parasite DNA in immune mice compared to controls 10 days after conjunctival challenge (P < 0.05). Antigen-specific gamma interferon production by T cells was increased at least 100-fold in cells harvested from immune mice (P < 0.05). Mucosal secretions containing T. cruzi-specific secretory immunoglobulin A harvested from immune mice were shown to protect against mucosal parasite infection (P < 0.05), demonstrating that mucosal antibodies can play a role in T. cruzi immunity. This model provides an important tool for detailed studies of mucosal immunity necessary for the development of mucosal vaccines.

Enhancement of innate and cell-mediated immunity by antimycobacterial antibodies.

We investigated the ability of human antibodies induced by Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination to protect against mycobacterial infections. Serum samples containing mycobacterium-specific antibodies were obtained from volunteers who had received two intradermal BCG vaccinations 6 months apart. Significant increases in lipoarabinomannan (LAM)-specific immunoglobulin G (IgG) were detected after both the primary and booster vaccinations. Effects of mycobacterium-specific antibodies on surface binding and internalization of BCG by neutrophils and monocytes/macrophages were studied, using green fluorescent protein (gfp)-expressing BCG. Surface-bound gfp-expressing BCG were distinguished from intracellular BCG by surface labeling with LAM-specific monoclonal antibody. Internalization of BCG by phagocytic cells was shown to be significantly enhanced in postvaccination serum samples. Furthermore, the inhibitory effects of neutrophils and monocytes/macrophages on mycobacterial growth were significantly enhanced by BCG-induced antibodies. The growth-inhibiting effects of postvaccination sera were reversed by preabsorption of IgG with Protein G. Finally, the helper effects of antimycobacterial antibodies for the induction of cell-mediated immune responses were investigated. BCG-induced antibodies significantly enhanced proliferation and gamma interferon production in mycobacterium-specific CD4(+) and CD8(+) T cells, as well as the proportion of proliferating and degranulating CD8(+) T cells. We conclude that mycobacterium-specific antibodies are capable of enhancing both innate and cell-mediated immune responses to mycobacteria.

Differential interleukin-8 and nitric oxide production in epithelial cells induced by mucosally invasive and noninvasive Trypanosoma cruzi trypomastigotes.

Trypanosoma cruzi metacyclic trypomastigotes (MT), but not blood form trypomastigotes (BFT), are highly mucosally infective. We investigated the abilities of MT and BFT to induce inflammation and/or intracellular killing activity within mucosal epithelia. BFT, but not MT, induced marked increases in interleukin-8, GRO-alpha, MCP-1, and nitric oxide production in HeLa and AGS cells, despite similar infectivities. MT may avoid induction of inflammation as an important biological mechanism facilitating mucosal invasion.

Differential effects of control and antigen-specific T cells on intracellular mycobacterial growth.

We investigated the effects of peripheral blood mononuclear cells expanded with irrelevant control and mycobacterial antigens on the intracellular growth of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in human macrophages. More than 90% of the cells present after 1 week of in vitro expansion were CD3(+). T cells were expanded from purified protein derivative-negative controls, persons with latent tuberculosis, and BCG-vaccinated individuals. T cells expanded with nonmycobacterial antigens enhanced the intracellular growth of BCG in suboptimal cultures of macrophages. T cells expanded with live BCG or lysates of Mycobacterium tuberculosis directly inhibited intracellular BCG. Recent intradermal BCG vaccination significantly enhanced the inhibitory activity of T cells expanded with mycobacterial antigens (P < 0.02), consistent with the induction of memory-immune inhibitory T-cell responses. Selected mycobacterial antigens (Mtb41 > lipoarabinomannan > 38kd > Ag85B > Mtb39) expanded inhibitory T cells, demonstrating the involvement of antigen-specific T cells in intracellular BCG inhibition. We studied the T-cell subsets and molecular mechanisms involved in the memory-immune inhibition of intracellular BCG. Mycobacteria-specific gammadelta T cells were the most potent inhibitors of intracellular BCG growth. Direct contact between T cells and macrophages was necessary for the BCG growth-enhancing and inhibitory activities mediated by control and mycobacteria-specific T cells, respectively. Increases in tumor necrosis factor alpha, interleukin-6, transforming growth factor beta, and vascular endothelial growth factor mRNA expression were associated with the enhancement of intracellular BCG growth. Increases in gamma interferon, FAS, FAS ligand, perforin, granzyme, and granulysin mRNA expression were associated with intracellular BCG inhibition. These culture systems provide in vitro models for studying the opposing T-cell mechanisms involved in mycobacterial survival and protective host immunity.

Type 1 immunity provides optimal protection against both mucosal and systemic Trypanosoma cruzi challenges.

Chagas' disease results from infection with Trypanosoma cruzi, a protozoan parasite that establishes systemic intracellular infection after mucosal invasion. We hypothesized that ideal vaccines for mucosally invasive, intracellular pathogens like T. cruzi should induce mucosal type 2 immunity for optimal induction of protective secretory immunoglobulin A (IgA) and systemic type 1 immunity protective against intracellular replication. However, differential mucosal and systemic immune memory could be difficult to induce because of reciprocal inhibitory actions between type 1 and type 2 responses. To test our hypotheses, we investigated the protective effects of type 1 and type 2 biased vaccines against mucosal and systemic T. cruzi challenges. Intranasal vaccinations were given with recombinant interleukin-12 (IL-12)- and IL-4-neutralizing antibody (Ab) for type 1 immune bias, or recombinant IL-4 and gamma interferon-neutralizing Ab for type 2 immune bias. Cytokine RNA and protein studies confirmed that highly polarized memory immune responses were induced by our vaccination protocols. Survival after virulent subcutaneous T. cruzi challenge was used to assess systemic protection. Mucosal protection was assessed by measuring the relative inhibition of parasite replication in mucosal tissues early after oral T. cruzi challenge, using both PCR and quantitative culture techniques. As expected, only type 1 responses protected against systemic challenges (P < 0.01). However, contrary to our original hypothesis, type 1 responses optimally protected against mucosal challenges as well (P < 0.05). Type 1 and type 2 biased vaccines induced similar secretory IgA responses. We conclude that future vaccines for T. cruzi and possibly other mucosally invasive, intracellular pathogens should induce both mucosal and systemic type 1 immunity.

Canarypox vaccines induce antigen-specific human gammadelta T cells capable of interferon-gamma production.

Induction of human gammadelta T cells was investigated in subjects who were vaccinated with live recombinant canarypox virus expressing human immunodeficiency virus (HIV) proteins or soluble MN rgp120. Both canarypox and rgp120 induced antigen-specific lymphoproliferative and interferon (IFN)-gamma responses. However, only canarypox vaccination induced increased gammadelta T cell responses detectable after secondary in vitro expansion (P<.02). These enhanced gammadelta T cell responses were specific for canarypox but not HIV antigens. Canarypox-specific gammadelta T cells were predominantly Vgamma9(+) and produced intracellular and secreted IFN-gamma. gammadelta T cell lines generated from canarypox vaccinees responded to canarypox antigens but not to mycobacterial antigens shown previously to induce bacille Calmette-Guérin-specific gammadelta T cells. Furthermore, canarypox vaccinations were associated with significantly higher NK cell expansions (P=.02). Increased IFN-gamma production by gammadelta T and NK cells could enhance the induction of protective type 1 memory immunity. Thus, stimulation of gammadelta T cells might be an important feature of live vaccines.

Immunoregulatory role of secreted glycoprotein G from respiratory syncytial virus.

The secretory glycoprotein (Gs) of respiratory syncytial virus (RSV) was enriched and investigated for its effects on T cells specific for RSV and unrelated antigens. Gs exhibited a dose dependent suppression of lymphoproliferative responses in peripheral blood mononuclear cells (PBMCs), specific for mycobacterial lysates or tetanus toxoid. However, Gs did not inhibit live RSV specific T cell responses. These results suggest that Gs may suppress immune response to unrelated antigens, but should not interfere with the overall development of RSV specific immunity.

Mucosal bacille calmette-Guérin vaccination of humans inhibits delayed-type hypersensitivity to purified protein derivative but induces mycobacteria-specific interferon-gamma responses.

We conducted a placebo-controlled, double-dose-escalation trial of oral bacille Calmette-Guérin (BCG) vaccination in 48 healthy volunteers. Seven of 32 BCG recipients became purified protein derivative (PPD)-positive after dose 1, and only 1 remained positive after dose 2, which suggests that oral BCG has inhibitory effects on delayed-type hypersensitivity (DTH) responses. Ten of the original placebo recipients and 11 oral BCG recipients were recruited to return for an intradermal BCG booster vaccination. Five of 10 original placebo recipients developed PPD responses >/=10 mm, but none of 11 oral BCG recipients developed PPD induration after they received an intradermal BCG booster (P<.05; Fisher's exact test). These results document persistent inhibitory effects of oral BCG vaccination on mycobacteria-specific DTH responses. Despite inhibition of DTH, oral BCG induced significant increases in mycobacteria-specific interferon (IFN)-gamma responses in peripheral blood mononuclear cells. More detailed studies of cytokine and homing molecule expression indicated that differential mucosal versus cutaneous trafficking may explain the dissociation between IFN-gamma and DTH responses.

In vitro measurement of protective mycobacterial immunity: antigen-specific expansion of T cells capable of inhibiting intracellular growth of bacille Calmette-Guérin.

We investigated the ability of T cells expanded with mycobacterial antigens from healthy purified protein derivative-reactive donors and bacille Calmette-Guérin (BCG)-vaccinated volunteers to inhibit intracellular growth of BCG. Peripheral blood mononuclear cells were incubated for 7 days with mycobacterial whole lysate, live BCG, tetanus toxoid as control antigen, or medium alone. Autologous monocytes were separated by plastic adherence, allowed to mature for 6 days, and infected with BCG before serving as target cells. Expanded effector cells were cocultured with target cells for 72 h. Cocultures were then treated with 0.2% saponin to lyse infected monocytes and release intracellular BCG. Quantities of viable BCG present in these lysates were studied by colony-forming unit counting and radiometric labeling. We reproducibly found that lymphocytes expanded with mycobacterial whole lysate or live BCG significantly inhibited the intracellular growth of BCG, compared with lymphocytes expanded with tetanus toxoid or rested in medium. In addition, BCG vaccination enhanced the ability of T cells to inhibit intracellular mycobacterial growth in 3 of 5 volunteers. This assay may be useful for estimates of protective immunity induced by tuberculosis vaccines in human trials.

Involvement of CD4(+) Th1 cells in systemic immunity protective against primary and secondary challenges with Trypanosoma cruzi.

In general, gamma interferon (IFN-gamma)-producing CD4(+) Th1 cells are important for the immunological control of intracellular pathogens. We previously demonstrated an association between parasite-specific induction of IFN-gamma responses and resistance to the intracellular protozoan Trypanosoma cruzi. To investigate a potential causal relationship between Th1 responses and T. cruzi resistance, we studied the ability of Th1 cells to protect susceptible BALB/c mice against virulent parasite challenges. We developed immunization protocols capable of inducing polarized Th1 and Th2 responses in vivo. Induction of parasite-specific Th1 responses, but not Th2 responses, protected BALB/c mice against virulent T. cruzi challenges. We generated T. cruzi-specific CD4(+) Th1 and Th2 cell lines from BALB/c mice that were activated by infected macrophages to produce their corresponding cytokine response profiles. Th1 cells, but not Th2 cells, induced nitric oxide production and inhibited intracellular parasite replication in T. cruzi-infected macrophages. Despite the ability to inhibit parasite replication in vitro, Th1 cells alone could not adoptively transfer protection against T. cruzi to SCID mice. In addition, despite the fact that the adoptive transfer of CD4(+) T lymphocytes was shown to be necessary for the development of immunity protective against primary T. cruzi infection in our SCID mouse model, protective secondary effector functions could be transferred to SCID mice from memory-immune BALB/c mice in the absence of CD4(+) T lymphocytes. These results indicate that, although CD4(+) Th1 cells can directly inhibit intracellular parasite replication, a more important role for these cells in T. cruzi systemic immunity may be to provide helper activity for the development of other effector functions protective in vivo.

A double-blind, placebo-controlled study of Mycobacterium-specific human immune responses induced by intradermal bacille Calmette-Guérin vaccination.

Recent studies have indicated that type 1 T cell responses (potent interferon-gamma and cytolytic responses, with absence of interleukin-4 production) are important for protective immunity against mycobacteria. These observations suggest that assays of type 1 T cell responses may be useful as surrogate markers of protective immunity in the evaluation of new tuberculosis vaccines. To be useful as surrogate markers, immunologic assays must distinguish between vaccine recipients and control subjects in clinical trials. Previous studies have shown that bacille Calmette-Guérin (BCG) vaccination can induce human type 1 T cell responses, but randomized trials have not been done to determine whether measurement of these responses can distinguish between BCG recipients and control subjects. We have conducted a double-blind, placebo-controlled trial of intradermal vaccination with two different BCG strains. We compared the mean lymphoproliferative, cytotoxic, Th1 and Th2 cytokine, and antibody responses detected in BCG and placebo recipients. These studies demonstrated that significant increases in Mycobacterium-specific T cell proliferative responses and type 1 cytokine responses were induced by BCG when compared with results with a placebo. In addition, BCG induced significant increases in Mycobacterium-specific antibody responses with an isotype profile characteristic of a type 1 cytokine bias. T cell and antibody assays involving the use of mycobacterial whole cell lysates or live BCG were able to discriminate between BCG and placebo recipients better than were assays using mycobacterial culture filtrates. These studies provide important information for the development of immunologic assays that might be useful as surrogate markers of protective immunity in future trials of new tuberculosis vaccines.

Persistence and boosting of bacille Calmette-Guérin-induced delayed-type hypersensitivity.

BACKGROUND: Bacille Calmette-Guérin (BCG) vaccination may induce persistent and booster purified protein derivative (PPD) responses that complicate tuberculosis screening efforts. OBJECTIVES: To investigate the effects of BCG vaccination on serial PPD tests and to study correlations between persistent delayed-type hypersensitivity and other potential surrogate markers of protective immunity. DESIGN: Cohort study. SETTING: Midwestern urban university. PARTICIPANTS: 69 healthy adults. INTERVENTIONS: BCG vaccination, blood samples drawn for immunologic studies, and PPD tests done sequentially over 1 to 3 years. MEASUREMENTS: Serial PPD induration, lymphoproliferation, and interferon-gamma responses. RESULTS: 10% of participants (95% CI, 4% to 20%) had persistent PPD responses of 15 mm or greater, and 3% (CI, 0% to 10%) demonstrated PPD boosting of 15 mm or greater 1 to 3 years after BCG vaccination. Intradermal BCG vaccination induced a larger number of persistent responses that were 10 mm or greater than did percutaneous BCG vaccination (12 of 46 participants compared with 1 of 23 participants; P = 0.05). Persistent and boosted delayed-type hypersensitivity correlated with mycobacterial-specific lymphoproliferation and interferon-gamma responses. CONCLUSIONS: Previous BCG vaccination reduces the predictive value of serial PPD testing. The lowest PPD predictive values will occur in persons without known tuberculosis exposure who were vaccinated recently or many times with intradermal BCG. In addition, BCG-related persistence and boosting of delayed-type hypersensitivity responses correlate with other potential surrogate markers of protective mycobacterial immunity.

Clinical reactogenicity of intradermal bacille Calmette-Guérin vaccination.

Clinical, microbiological, and immunologic responses were evaluated in volunteers vaccinated intradermally with bacille Calmette-Guérin (BCG). Most volunteers (98%) developed ulcerative lesions that drained for a mean +/- SE of 4.3 +/- 0.29 weeks. Mycobacterial DNA was detected by a polymerase chain reaction-based amplification technique in biopsy specimens from BCG ulcers 2 weeks after vaccination and in blood specimens 3 days after vaccination. Mycobacteria were cultured from ulcer drainage 2 months after vaccination, demonstrating a prolonged potential risk of contact spread of the vaccine strain. The duration of ulcer drainage was inversely correlated with prevaccination lymphoproliferative (r = -0.515; P < .002) and interferon gamma (r = -0.841; P < .002) responses specific to mycobacteria and directly correlated with postvaccination increases in lymphoproliferative (r = 0.498; P < .002) and interferon gamma (r = 0.688; P < .02) responses specific to mycobacteria. These results demonstrate the clinical reactogenicity of BCG and the potential risk of contact spread of the vaccine strain and suggest that clinical reactogenicity is a trade-off for the induction of protective mycobacterial immunity.

Bacille Calmette-Guérin vaccination enhances human gamma delta T cell responsiveness to mycobacteria suggestive of a memory-like phenotype.

Bacille Calmette-Guérin (BCG) immunity can be studied as one experimental model for mycobacterial protective immunity. We have used flow cytometry to investigate human T cell subsets induced by BCG vaccination. PBMC harvested from BCG-vaccinated individuals and controls were stimulated with mycobacterial Ags, and the T cell subsets present after 7 days of in vitro expansion were characterized. The most dramatic expansions induced by mycobacterial Ags were detected in gamma delta T cells. The gamma delta T cell expansions measured after in vitro stimulation with mycobacterial Ags were significantly greater in BCG responders compared with nonsensitized controls, indicating that BCG vaccination induced gamma delta T cell activation associated with enhanced secondary responses. The majority of gamma delta T cells induced by BCG vaccination were gamma 9+ delta 2+ T cells reactive with isoprenyl pyrophosphates. Coculture with CD4+ T cells induced optimal gamma delta T cell expansion, although IL-2 alone could provide this helper function in the absence of CD4+ T cells. Gamma delta T cells were found to provide helper functions for mycobacterial specific CD4+ and CD8+ T cells as well, demonstrating reciprocal stimulatory interactions between gamma delta T cells and other T cell subsets. Finally, prominent mycobacterial specific gamma delta T cell expansions were detected in a subset of unvaccinated controls with evidence for prior sensitization to mycobacterial lysates (elevated mycobacterial specific lymphoproliferative responses). These latter findings are consistent with the hypothesis that exposure to atypical mycobacteria or related environmental Ags may induce gamma delta T cells cross-reactive with Ags present in the Mycobacterium tuberculosis complex. Our results suggest that gamma delta T cells may be capable of developing a memory immune-like phenotype, and therefore might be important targets for new vaccines.

Differential mucosal infectivity of different life stages of Trypanosoma cruzi.

Mucosal invasion is an important method of vector-borne transmission of Trypanosoma cruzi to human hosts. We previously have shown that low numbers of virulent insect-derived metacyclic trypomastigotes (IMT) collected from the excreta of reduviid bugs were highly efficient in infecting mice through gastrointestinal and conjunctival mucosa. However, we have recently found that blood-form trypomastigotes (BFT) of T. cruzi cannot efficiently initiate mucosal infection after an oral challenge of the gastrointestinal tract. Evidence for systemic infection after oral challenge with BFT was sought by microscopic parasitemia examinations of fresh blood, polymerase chain reaction analyses with DNA extracted from mouse blood using primers specific for a nuclear repeat present in the T. cruzi genome, and by Western blots of parasite lysates probed with individual mouse serum. Oral challenge doses of 1,000-10,000 BFT were found to rarely initiate mucosal infection. In contrast, 1,000 IMT delivered orally was a sufficient challenge for the consistent infection of 100% of control BALB/c mice. The exceptions infected mucosally by BFT involved animals with mucosal defects due to trauma or ulcerative/periodontal diseases. These data suggest that IMT have uniquely specialized functions for mucosal invasion that are not normally present in BFT.

Gastric invasion by Trypanosoma cruzi and induction of protective mucosal immune responses.

Trypanosoma cruzi is an intracellular parasite transmitted from a reduviid insect vector to humans by exposure of mucosal surfaces to infected insect excreta. We have used an oral challenge murine model that mimics vector-borne transmission to study T. cruzi mucosal infection. Although gastric secretions have microbicidal activity against most infectious pathogens, we demonstrate that T. cruzi can invade and replicate in the gastric mucosal epithelium. In addition, gastric mucosal invasion appears to be the unique portal of entry for systemic T. cruzi infection after oral challenge. The mucosal immune responses stimulated by T. cruzi gastric infection are protective against a secondary mucosal parasite challenge. This protective mucosal immunity is associated with increased numbers of lymphocytes that secrete parasite-specific immunoglobulin A. Our results document the first example of systemic microbial invasion through gastric mucosa and suggest the feasibility of a mucosal vaccine designed to prevent infection with this important human pathogen.

Immune responses stimulated by percutaneous and intradermal bacille Calmette-Guérin.

Healthy volunteers were randomized to receive percutaneous or intradermal bacille Calmette-Guérin (BCG) vaccination. Delayed-type hypersensitivity (DTH) to tuberculin, as well as proliferative and interferon-gamma responses in peripheral blood mononuclear cells stimulated by whole cell lysates and culture filtrates of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare, were compared before and after vaccination. Positive DTH reactions were detected in 83% of intradermal and 40% of percutaneous BCG recipients 3 months after vaccination (P < .004). M. tuberculosis-specific proliferation was increased after intradermal BCG (P < .01) but not after percutaneous BCG compared with prevaccination responses. In addition, M. tuberculosis-specific interferon-gamma production was increased after intradermal BCG compared with both prevaccination responses (P < .04) and those measured after percutaneous BCG (P < .05). Predominant immune responses stimulated by BCG were directed against antigens present in mycobacterial whole cell lysate. These results indicate that the BCG vaccination route can affect both in vivo and in vitro immune responses.