Subcutaneous Administration of a 10-Fold-Lower Dose of a Commercial Human Tuberculosis Vaccine,Mycobacterium bovis Bacillus Calmette-Guérin Danish,Induced Levels of Protection against Bovine Tuberculosis and Responses in the Tuberculin Intradermal Test Similar to Those Induced by a Standard Cattle Dose

Vaccination of cattle with a commercial human tuberculosis (TB) vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG) Danish, at a dose equivalent to 5 human doses of BCG has protected these animals against TB in field and experimental trials. There is interest in determining whether a 10-fold-lower dose could still protect cattle but not induce a tuberculin intradermal test response. Two groups of calves (n = 9/group) were vaccinated subcutaneously with a lyophilized BCG Danish vaccine containing either 0.5 (1 × 10⁵ to 4 × 10⁵ CFU) or 5 (1 × 10⁶ to 4 × 10⁶ CFU) human doses of BCG Danish, with an additional group of 10 calves serving as nonvaccinated controls. Fifteen weeks after vaccination, these animals were challenged intratracheally with 5 × 10³ CFU of virulent M. bovis and another 15 weeks later were slaughtered and examined for the presence of tuberculous lesions. Vaccination of the calves with either 0.5 or 5 equivalent human doses of BCG Danish induced similar levels of protection against challenge with M. bovis, with both groups showing significant reductions in the pathological and microbiological parameters compared to those for the the control group (P < 0.05). Vaccination with either of the two BCG doses induced similar numbers of animals responding to the tuberculin intradermal test at 11 weeks postvaccination. Vaccination with a 0.5 equivalent human dose of a commercial lyophilized BCG vaccine can protect cattle against challenge with M. bovis.     

Novel Recombinant BCG Coexpressing Ag85B, ESAT-6 and Rv2608 Elicits Significantly Enhanced Cellular Immune and Antibody Responses in C57BL/6 Mice

Tuberculosis (TB) remains an enormous global health problem, and a new vaccine against TB more potent than the current inadequate vaccine, the Bacille Calmette‐Guérin (BCG), is urgently needed. BCG has proven to be an effective recombinant delivery vehicle for foreign antigens because of its ability to induce long‐lived specific humoral and cellular immunity. Experimental evidences have revealed that Ag85B, ESAT‐6 and Rv2608 are important immunodominant antigens of Mycobacterium tuberculosis and are all promising vaccine candidate molecules. In this study, we have constructed a novel recombinant BCG (rBCG) expressing fusion protein Ag85B‐ESAT6‐Rv2608 and evaluated the immunogenicity of rBCG in C57BL/6 mice. Results show there is strong TB‐specific CD4⁺ and CD8⁺ T lymphocytes proliferative response in mice immunized with rBCG vaccine, especially the cytotoxic CD8⁺ T cells playing an important role in protection against TB. And rBCG immunization has induced a significantly strong Th1 immune response, characterized by the increased ratio of IgG2b/IgG1. Results also show that rBCG immunization could increase the secretion of Th1 cytokines such as TNF‐α and IL‐2 and could decrease the secretion of Th2 cytokine IL‐10. Moreover, it was shown that rBCG immunization induced a strong humoral response in mice, characterized by the elevated IgG titre. Therefore, we conclude that this rBCG immunization could increase both cellular immune response and antigen‐specific humoral response significantly as compared to BCG immunization in mice. The above results illustrated that rBCG::Ag85B‐ESAT6‐Rv2608 is a potential candidate against M. tuberculosis for further study.     

Recombinant BCG coexpressing Ag85B,ESAT-6 and Rv3620c elicits specific Th1 immune responses in C57BL/6 mice

Tuberculosis (TB) remains to be an enormous global health problem. The inconsistent protection efficacy of Bacille Calmette-Guérin (BCG) calls for new vaccines for TB. One choice to improve the efficacy of BCG vaccine is recombinant BCG (rBCG). Experimental evidences have revealed that Ag85B, ESAT-6 and Rv3620c are important immunodominant antigens of Mycobacterium tuberculosis. In this study, we have constructed a novel rBCG expressing fusion protein Ag85B-ESAT6-Rv3620c and evaluated the immunogenicity of this rBCG in C57BL/6 mice. Results show that there is a strong TB-specific CD4⁺ and CD8⁺ T lymphocytes proliferation in mice immunized with this rBCG vaccine. A single dose immunization of rBCG could induce a significantly strong Th1 immune response characterized by an increasing ratio of antigen-specific IgG2b/IgG1 as well as a high expression level of Th1 cytokines such as IFN-γ, TNF-α and IL-2. This conclusion was confirmed by a decreased secretion of Th2 cytokine IL-10. Moreover, this rBCG induced a strong humoral response in mice with an increasing antigen-specific IgG titer. Therefore, we concluded that this rBCG could significantly increase both Th1 type cellular immune response and antigen-specific humoral response compared with BCG. The above observations demonstrated that rBCG::Ag85B-ESAT6-Rv3620c is a potential candidate vaccine against M. tuberculosis for further study.     

A Replication-Limited Recombinant Mycobacterium bovis BCG vaccine against tuberculosis designed for human immunodeficiency virus-positive persons is safer and more efficacious than BCG

Tuberculosis is the leading cause of death in AIDS patients, yet the current tuberculosis vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is contraindicated for immunocompromised individuals, including human immunodeficiency virus-positive persons, because it can cause disseminated disease; moreover, its efficacy is suboptimal. To address these problems, we have engineered BCG mutants that grow normally in vitro in the presence of a supplement, are preloadable with supplement to allow limited growth in vivo, and express the highly immunoprotective Mycobacterium tuberculosis 30-kDa major secretory protein. The limited replication in vivo renders these vaccines safer than BCG in SCID mice yet is sufficient to induce potent cell-mediated and protective immunity in the outbred guinea pig model of pulmonary tuberculosis. In the case of one vaccine, rBCG(mbtB)30, protection was superior to that with BCG (0.3-log fewer CFU of M. tuberculosis in the lung [P < 0.04] and 0.6-log fewer CFU in the spleen [P = 0.001] in aerosol-challenged animals [means for three experiments]); hence, rBCG(mbtB)30 is the first live mycobacterial vaccine that is both more attenuated than BCG in the SCID mouse and more potent than BCG in the guinea pig. Our study demonstrates the feasibility of developing safer and more potent vaccines against tuberculosis. The novel approach of engineering a replication-limited vaccine expressing a recombinant immunoprotective antigen and preloading it with a required nutrient, such as iron, that is capable of being stored should be generally applicable to other live vaccine vectors targeting intracellular pathogens.     

Protection by a Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Vaccine Expressing Shiga Toxin 2 B Subunit against Shiga Toxin-Producing Escherichia coli in Mice

We have developed a novel vaccine against Shiga toxin (Stx)-producing Escherichia coli (STEC) infection using a recombinant Mycobacterium bovis BCG (rBCG) system. Two intraperitoneal vaccinations with rBCG expressing the Stx2 B subunit (Stx2B) resulted in an increase of protective serum IgG and mucosal IgA responses to Stx2B in BALB/c mice. When orally challenged with 10³ CFU of STEC strain B2F1 (O91: H21), the immunized mice survived statistically significantly longer than the nonvaccinated mice. We suggest that intraperitoneal immunization with rBCG expressing Stx2B would be a potential vaccine strategy for STEC.     

Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Vectors Prime for Strong Cellular Responses to Simian Immunodeficiency Virus Gag in Rhesus Macaques

Live attenuated nonpathogenic Mycobacterium bovis bacillus Calmette-Guérin (BCG) mediates long-lasting immune responses, has been safely administered as a tuberculosis vaccine to billions of humans, and is affordable to produce as a vaccine vector. These characteristics make it very attractive as a human immunodeficiency virus (HIV) vaccine vector candidate. Here, we assessed the immunogenicity of recombinant BCG (rBCG) constructs with different simian immunodeficiency virus (SIV)gag expression cassettes as priming agents followed by a recombinant replication-incompetent New York vaccinia virus (NYVAC) boost in rhesus macaques. Unmutated rBCG constructs were used in comparison to mutants with gene deletions identified in an in vitro screen for augmented immunogenicity. We demonstrated that BCG-SIVgag is able to elicit robust transgene-specific priming responses, resulting in strong SIV epitope-specific cellular immune responses. While enhanced immunogenicity was sustained at moderate levels for >1 year following the heterologous boost vaccination, we were unable to demonstrate a protective effect after repeated rectal mucosal challenges with pathogenic SIVmac251. Our findings highlight the potential for rBCG vaccines to stimulate effective cross-priming and enhanced major histocompatibility complex class I presentation, suggesting that combining this approach with other immunogens may contribute to the development of effective vaccine regimens against HIV.     

Immunogenicity and Protective Efficacy Conferred by a Novel Recombinant Mycobacterium bovis Bacillus Calmette‐Guérin Strain Expressing Interleukin‐12p70 of Human Cytokine and Ag85A of Mycobacterium tuberculosis Fusion Protein

Mycobacterium bovis bacillus Calmette‐Guérin (BCG) immunization provides protection against tuberculosis (TB) in infants, but the antituberculosis protective immunity wanes gradually after initial immunization and lasts less than 15 years. Therefore, more efficacious vaccines are urgently needed. In this study, we constructed a new tuberculosis vaccine of recombinant BCG strain (rBCG‐IA), which could express IL‐12p70 of human cytokine and Ag85A of M. tuberculosis fusion protein, and investigated its immunogenicity in BALB/c mice by measuring antibody titres, proliferation rate of splenocytes, ratios of CD4⁺ T and CD8⁺ T cells stimulated by specific antigens and levels of IFN‐γ production in antigen‐stimulated splenocyte cultures. Meanwhile, we evaluated its protective efficacy against M. tuberculosis H37Rv infection through detecting lung histopathology, organ bacterial loads and lung acid‐fast stain. Immunogenicity experiments illustrated that from 2nd to 8th week after immunization, the rBCG‐IA vaccine was able to induce the highest level of antibody titres, proliferation rate of splenocytes and IFN‐γ production among groups and gained improved ratio of CD4⁺ T and CD8⁺ T cells from 6th to 8th week after vaccination. And from 2nd to 8th week after M. tuberculosis H37Rv infection, the score of pathology and bacterial loads in the rBCG‐IA group were obviously lower than that in rBCG‐I group, rBCG‐A group or control group (PBST group), but similar to that in BCG group. This study suggested that rBCG‐IA was able to elicit stronger humoral and cellular immune responses, but could only confer similar protective efficacy compared with its parental BCG vaccine.     

MHC‐restricted Ag85B‐specific CD8+ T cells are enhanced by recombinant BCG prime and DNA boost immunization in mice

Despite efforts to develop effective treatments and vaccines, Mycobacterium tuberculosis (Mtb), particularly pulmonary Mtb, continues to provide major health challenges worldwide. To improve immunization against the persistent health challenge of Mtb infection, we have studied the CD8⁺ T cell response to Bacillus Calmette‐Guérin (BCG) and recombinant BCG (rBCG) in mice. Here, we generated CD8⁺ T cells with an rBCG‐based vaccine encoding the Ag85B protein of M. kansasii, termed rBCG‐Mkan85B, followed by boosting with plasmid DNA expressing the Ag85B gene (DNA‐Mkan85B). We identified two MHC‐I (H2‐K^d)‐restricted epitopes that induce cross‐reactive responses to Mtb and other related mycobacteria in both BALB/c (H2^d) and CB6F1 (H2^b/d) mice. The H2‐K^d‐restricted peptide epitopes elicited polyfunctional CD8⁺ T cell responses that were also highly cross‐reactive with those of other proteins of the Ag85 complex. Tetramer staining indicated that the two H2‐K^d‐restricted epitopes elicit distinct CD8⁺ T cell populations, a result explained by the X‐ray structure of the two peptide/H2‐K^d complexes. These results suggest that rBCG‐Mkan85B vector‐based immunization and DNA‐Mkan85B boost may enhance CD8⁺ T cell response to Mtb, and might help to overcome the limited effectiveness of the current BCG in eliciting tuberculosis immunity.     

Recombinant Mycobacterium bovis BCG Expressing the Chimeric Protein of Antigen 85B and ESAT-6 Enhances the Th1 Cell-Mediated Response

The chimeric protein that relies on the T-cell epitopes of antigen 85B (Ag85B) and the 6-kDa early secreted antigen target (ESAT-6) has been demonstrated to augment the Th1 immune response. In this study, we developed a recombinant Mycobacterium bovis BCG (rBCG) strain that secretes the chimeric protein of Ag85B and ESAT-6 (rBCG-A_N-E-A_C). Immunization with this rBCG strain induced stronger antigen-specific gamma interferon (IFN-γ) activities, as determined by an enzyme-linked immunospot assay, and higher levels of antigen-specific CD4⁺ and CD8⁺ T-cell responses than those in the control groups immunized with either rBCG expressing the Ag85B-ESAT-6 fusion protein (rBCG-A-E) or BCG. Likewise, rBCG-A_N-E-A_C significantly increased the level of production of the major Th1 cytokines IFN-γ and tumor necrosis factor alpha in splenocyte cultures to levels comparable to those elicited by control BCG. Moreover, the antigen-specific immunoglobulin 2c (IgG2c)/IgG1 ratio for mice immunized with rBCG-A_N-E-A_C was also much higher than the ratios for the other immunized groups. Together, these results indicate that this rBCG-A_N-E-A_C strain enhances the Th1 cell-mediated response and may serve as a potential vaccine against M. tuberculosis.Mycobacterium bovis bacillus Calmette Guérin (BCG) is the only vaccine against tuberculosis (TB) currently available and exhibits various levels of efficacy for the prevention of pulmonary TB (range, 0 to 80%) in different trials (9). BCG has a protective effect in children, particularly against tuberculous meningitis; however, it does not satisfactorily prevent the development of pulmonary TB in adults and fails to protect individuals against reinfection (1). Given the rate of mortality from TB worldwide, with more 8 million new cases and 2 million deaths occurring annually (2), newer strategies need to be implemented to improve BCG or vaccines more effective than BCG urgently need to be developed.One approach that might be used to increase the efficacy of BCG could be to construct a recombinant BCG (rBCG) which either overexpresses immunogenic antigens or modulates the ensuing immune response (8). rBCG vaccines are attractive because of the widespread experience with their use, the known immunogenicity associated with protection against the worst forms of the disease in children, and the safety profiles of standard BCG strains (13). Two rBCG vaccines have been entered into clinical trials. This includes rBCG30, which expresses the antigen 85B (Ag85B) protein, and ΔureC hly-positive rBCG, which expresses listeriolysin and which is urease deficient (12, 15). It is hoped that these vaccines will provide a strong and perhaps longer-lasting immune response than that achieved with the conventional BCG vaccine.The most effective defined-antigen TB vaccines will likely require the induction of both cell-mediated and humoral immune responses. Ag85B and the 6-kDa early secreted antigen target (ESAT-6) have been identified as two of the most promising vaccine candidates which are strongly recognized by T lymphocytes (3, 19). In a previous study, we relied on the T-cell epitopes of Ag85B and ESAT-6 to design a chimeric protein by inserting ESAT-6 into Ag85B from amino acids 167 to 182 and demonstrated that this recombination of Ag85B and ESAT-6 could improve the immunogenicity and enhance the T-helper type 1 (Th1) cell-mediated immune response (27). This finding prompted us to explore further the efficacy of rBCG overexpressing this chimeric protein. In this study, we constructed rBCG expressing chimeric protein Ag85B_N-ESAT-6-Ag85B_C (rBCG-A_N-E-A_C) and further compared the immune response to that protein with that to rBCG expressing the Ag85B-ESAT-6 fusion protein (rBCG-A-E) and BCG.     

Recombinant Mycobacterium bovis BCG is a promising platform to develop vaccines against Trypansoma cruzi infection

Chagas disease, caused by the hemoflagelate parasite Trypanosoma cruzi, is one of the most prevalent endemic parasitoses, affecting 7–8 million people. Due to the complexity of the infection, no vaccines are available at present. The extraordinary adjuvant capacity of bacille Calmette–Guérin (BCG) was explored in this work to develop a vaccine candidate to protect against T. cruzi infection using the recombinant BCG (rBCG) vaccine platform. Three antigens of the parasite corresponding to the N and C terminal fragments of the enzyme trans-sialidase (NT-TS and CT-TS, respectively) and a fragment of the cruzipain enzyme (CZf) were cloned into the vectors pUS997 and pUS2000 and transformed into the BCG Pasteur strain. In vaccinated mice, rBCG expressing NT-TS in pUS2000 plasmid provided the highest protection and the lowest parasitemia after challenging BALB/c mice with a 50% lethal dose of parasites. When mice vaccinated with pUS2000-NT-TS were challenged with a 100% lethal dose of parasite, high levels of protection were also obtained, together with a low degree of cardiac lesions 120 days after infection. In immunized mice with pUS2000-NT-TS/rBCG clone, the proliferation of CD4⁺ cells from splenocytes stimulated with the TS antigen was significant; this stimulation increased interferon (IFN)-γ and interleukin (IL)-17 within CD4⁺ T lymphocytes (LTCD4⁺) cells and IFN-γ and CD107 expression within LTCD8⁺ cells. Therefore, pUS2000-NT-TS/rBCG conferred high levels of protection, which correlated with an immune response orientated towards a T helper type 1 (Th1)/Th17 profile, together with an LTC-specific response, indicating that rBCG is a promising platform to develop vaccines against T. cruzi.     

Safety and Immunogenicity of Escalating Dosages of a Single Oral Administration of Peru-15 pCTB,a Candidate Live,Attenuated Vaccine against Enterotoxigenic Escherichia coli and Vibrio cholerae

Enterotoxigenic Escherichia coli (ETEC) organisms are a leading cause of infectious diarrhea in developing countries. A live, attenuated cholera strain that expresses high levels of the nontoxic B subunit of cholera toxin, which might also serve as an ETEC protective antigen, was evaluated for safety, excretion, and immunogenicity in healthy volunteers. We enrolled four inpatient dose-escalation cohorts of 15 to 16 eligible subjects to randomly (3:1) receive a single oral dose of vaccine or placebo (buffer alone), evaluating 1 ×10⁷, 1 ×10⁸, 1 ×10⁹, and 1 ×10¹⁰ CFU of the vaccine. The vaccine was well tolerated, although some subjects experienced moderate diarrhea. The serum Inaba vibriocidal antibody response appeared to display a dose-response relationship with increasing dosages of vaccine, plateauing at the 10⁹-CFU dosage. The serum antitoxin (cholera toxin and heat-labile enterotoxin) antibody seroconversion rate (4-fold increase over baseline) also appeared to display a dose-response relationship. The vaccine strain was excreted in stool cultures, displaying a dose-response relationship. A single oral dose of Peru-15 pCTB at dosages up to 1 ×10¹⁰ CFU was safe and immunogenic in this first-in-human trial. These encouraging data support the ongoing clinical development of this candidate combined cholera and ETEC vaccine. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT00654108","term_id":"NCT00654108"}}NCT00654108.)     

rBCG30-Induced Immunity and Cross-Protection against Mycobacterium leprae Challenge Are Enhanced by Boosting with the Mycobacterium tuberculosis 30-Kilodalton Antigen 85B

Leprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimal Mycobacterium bovis BCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG against Mycobacterium tuberculosis and Mycobacterium bovis challenge in animal models, for efficacy against Mycobacterium leprae challenge in a murine model of leprosy. rBCG30 overexpresses the M. tuberculosis 30-kDa major secretory protein antigen 85B, which is 85% homologous with the M. leprae homolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viable M. leprae into each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating the M. leprae bacteria per footpad. Both BCG and rBCG30 induced significant protection against M. leprae challenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purified M. tuberculosis or M. leprae antigen 85B also induced protection against M. leprae challenge but less so than BCG or rBCG30. Notably, boosting rBCG30 with M. tuberculosis antigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection against M. leprae challenge. Thus, rBCG30, a vaccine that induces improved protection against M. tuberculosis, induces cross-protection against M. leprae that is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy.     

Differential Mycobacterium bovis BCG Vaccine-Derived Efficacy in C3Heb/FeJ and C3H/HeOuJ Mice Exposed to a Clinical Strain of Mycobacterium tuberculosis

The global epidemic caused by the bacterial pathogen Mycobacterium tuberculosis continues unabated. Moreover, the only available vaccine against tuberculosis, Mycobacterium bovis bacillus Calmette-Guérin (BCG), demonstrates variable efficacy. To respond to this global threat, new animal models that mimic the pathological disease process in humans are required for vaccine testing. One new model, susceptible C3Heb/FeJ mice, is similar to human tuberculosis in that these animals are capable of forming necrotic tubercle granulomas, in contrast to resistant C3H/HeOuJ mice. In this study, we evaluated the impact of prior BCG vaccination of C3Heb/FeJ and C3H/HeOuJ mice on exposure to a low-dose aerosol of Mycobacterium tuberculosis W-Beijing strain SA161. Both BCG-vaccinated murine strains demonstrated reduced bacterial loads 25 days after infection compared to controls, indicating vaccine efficacy. However, during chronic infection, vaccine efficacy waned in C3H/HeOuJ but not in C3Heb/FeJ mice. Protection in vaccinated C3Heb/FeJ mice was associated with reduced numbers of CD11b⁺ Gr1⁺ cells, increased numbers of effector and memory T cells, and an absence of necrotic granulomas. BCG vaccine efficacy waned in C3H/HeOuJ mice, as indicated by reduced expression of gamma interferon (IFN-γ) and increased expressions of interleukin-17 (IL-17), IL-10, and Foxp3 by T cells compared to C3Heb/FeJ mice. This is the first murine vaccine model system described to date that can be utilized to dissect differential vaccine-derived immune efficacy.     

Differences in Response Among Inbred Mouse Strains to Infection with Small Doses of Mycobacterium bovis BCG

Intravenous infection of six inbred mouse strains with small doses of dispersed cells of Mycobacterium bovis BCG (15.5 × 10³ or 15.5 × 10⁴ colony-forming units) separated them into resistant (C3H/HeCr, A/J, and DBA/2) and sensitive (B10.A, C57BL/6, and BALB/c) strains as assessed by the magnitude of bacterial multiplication in the spleens at 28 days. The two groups were more sharply separated after infection with the lower dose of BCG (15.5 × 10³ colony-forming units), which allowed for true multiplication of the bacteria in the spleens of permissive hosts, expressed as the ratio of the number of BCG recovered from the spleens to the number of BCG injected. This coefficient of increase was less than 1 in resistant strains, whereas it was higher than 2.5 in sensitive strains. Significant splenomegaly developed only in mice of the sensitive strains infected with BCG when compared with uninfected controls. There was no correlation between the magnitude of the delayed-type hypersensitivity (DTH) to BCG and susceptibility to infection: DTH was absent in both the sensitive and the resistant strains when the smaller dose of BCG was used for infection. Moreover, significant DTH was detected in animals of the most sensitive (BALB/c) as well as of the most resistant (C3H/HeCr) strain when the higher dose of BCG (15.5 × 10⁴) was used for immunization. These results document significant genetic differences in the ability of inbred mice to inhibit bacterial multiplication after infection with small dispersed doses of BCG. Resistance to BCG multiplication, in this model, does not appear to be related to the establishment of DTH.     

Mycobacterium bovis BCG Vaccination Induces Divergent Proinflammatory or Regulatory T Cell Responses in Adults

Mycobacterium bovis bacillus Calmette-Guérin (BCG), the only currently available vaccine against tuberculosis, induces variable protection in adults. Immune correlates of protection are lacking, and analyses on cytokine-producing T cell subsets in protected versus unprotected cohorts have yielded inconsistent results. We studied the primary T cell response, both proinflammatory and regulatory T cell responses, induced by BCG vaccination in adults. Twelve healthy adult volunteers who were tuberculin skin test (TST) negative, QuantiFERON test (QFT) negative, and BCG naive were vaccinated with BCG and followed up prospectively. BCG vaccination induced an unexpectedly dichotomous immune response in this small, BCG-naive, young-adult cohort: BCG vaccination induced either gamma interferon-positive (IFN-γ⁺) interleukin 2-positive (IL-2⁺) tumor necrosis factor α-positive (TNF-α⁺) polyfunctional CD4⁺ T cells concurrent with CD4⁺ IL-17A⁺ and CD8⁺ IFN-γ⁺ T cells or, in contrast, virtually absent cytokine responses with induction of CD8⁺ regulatory T cells. Significant induction of polyfunctional CD4⁺ IFN-γ⁺ IL-2⁺ TNF-α⁺ T cells and IFN-γ production by peripheral blood mononuclear cells (PBMCs) was confined to individuals with strong immunization-induced local skin inflammation and increased serum C-reactive protein (CRP). Conversely, in individuals with mild inflammation, regulatory-like CD8⁺ T cells were uniquely induced. Thus, BCG vaccination either induced a broad proinflammatory T cell response with local inflammatory reactogenicity or, in contrast, a predominant CD8⁺ regulatory T cell response with mild local inflammation, poor cytokine induction, and absent polyfunctional CD4⁺ T cells. Further detailed fine mapping of the heterogeneous host response to BCG vaccination using classical and nonclassical immune markers will enhance our understanding of the mechanisms and determinants that underlie the induction of apparently opposite immune responses and how these impact the ability of BCG to induce protective immunity to TB.     

The role of Gr-1+ cells and tumour necrosis factor-α signalling during Clostridium difficile colitis in mice

The host response to Clostridium difficile infection in antibiotic-treated mice is characterized by robust recruitment of Gr-1⁺ cells, increased expression of inflammatory cytokines including tumour necrosis factor-α (TNF-α), and the development of severe epithelial damage. To investigate the role of Gr-1⁺ cells and TNF-α during C. difficile colitis, we treated infected mice with monoclonal antibodies against Gr-1 or TNF-α. Mice were challenged with vegetative cells of C. difficile strain VPI 10463 following treatment with the third-generation cephalosporin ceftriaxone. Ceftriaxone treatment alone was associated with significant changes in cytokine expression within the colonic mucosa but not overt inflammatory histopathological changes. In comparison, C. difficile infection following ceftriaxone treatment was associated with increased expression of inflammatory cytokines and chemokines including Cxcl1, Cxcl2, Il1b, Il17f and Tnfa, as well as robust recruitment of Ly6C^Mid Gr-1^High neutrophils and Ly6C^High Gr-1^Mid monocytes and the development of severe colonic histopathology. Anti-Gr-1 antibody treatment resulted in effective depletion of both Ly6C^Mid Gr-1^High neutrophils and Ly6C^High Gr-1^Mid monocytes: however, we observed no protection from the development of severe pathology or reduction in expression of the pro-inflammatory cytokines Il1b, Il6, Il33 and Tnfa following anti-Gr-1 treatment. By contrast, anti-TNF-α treatment did not affect Gr-1⁺ cell recruitment, but was associated with increased expression of Il6 and Il1b. Additionally, Ffar2, Ffar3, Tslp, Tff and Ang4 expression was significantly reduced in anti-TNF-α-treated animals, in association with marked intestinal histopathology. These studies raise the possibility that TNF-α may play a role in restraining inflammation and protecting the epithelium during C. difficile infection.     

Recombinant BCG coexpressing Ag85B, CFP10, and interleukin-12 induces multifunctional Th1 and memory T cells in mice

Mycobacterium tuberculosis (MTB) continues to be a leading cause of human deaths due to an infectious agent. Current efforts are focused on making better TB vaccines. We describe the generation and immunological characterization of recombinant BCG (rBCG). This rBCG was generated by incorporating an expression plasmid encoding two mycobacterial antigens (Ag85B and CFP10) and human interleukin (IL)-12 into a BCG strain. Immunogenicity studies in mice showed that rBCG coexpressing Ag85B, CFP10, and IL-12 (rBCG::Ag85B-CFP10-IL-12) induces a robust immune response in mice. The rBCG vaccine promotes a T-cell response against MTB that is characterized by a high proportion of polyfunctional and memory T cells in spleen and lung. Our results showed strong immunogenicity and mycobacterial growth inhibition of rBCG::Ag85B-CFP10 plus IL-12 than that of BCG vaccine.     

Characterization of Novel Multiantigenic Vaccine Candidates with Pan-HLA Coverage against Mycobacterium tuberculosis

The low protection by the bacillus Calmette-Guérin (BCG) vaccine and existence of drug-resistant strains require better anti-Mycobacterium tuberculosis vaccines with a broad, long-lasting, antigen-specific response. Using bioinformatics tools, we identified five 19- to 40-mer signal peptide (SP) domain vaccine candidates (VCs) derived from M. tuberculosis antigens. All VCs were predicted to have promiscuous binding to major histocompatibility complex (MHC) class I and II alleles in large geographic territories worldwide. Peripheral mononuclear cells (PBMC) from healthy naïve donors and tuberculosis patients exhibited strong proliferation that correlated positively with Th1 cytokine secretion only in healthy naïve donors. Proliferation to SP VCs was superior to that to antigen-matched control peptides with similar length and various MHC class I and II binding properties. T-cell lines induced to SP VCs from healthy naïve donors had increased CD44^high/CD62L⁺ activation/effector memory markers and gamma interferon (IFN-γ), but not interleukin-4 (IL-4), production in both CD4⁺ and CD8⁺ T-cell subpopulations. T-cell lines from healthy naïve donors and tuberculosis patients also manifested strong, dose-dependent, antigen-specific cytotoxicity against autologous VC-loaded or M. tuberculosis-infected macrophages. Lysis of M. tuberculosis-infected targets was accompanied by high IFN-γ secretion. Various combinations of these five VCs manifested synergic proliferation of PBMC from selected healthy naïve donors. Immunogenicity of the best three combinations, termed Mix1, Mix2, and Mix3 and consisting of 2 to 5 of the VCs, was then evaluated in mice. Each mixture manifested strong cytotoxicity against M. tuberculosis-infected macrophages, while Mix3 also manifested a VC-specific humoral immune response. Based on these results, we plan to evaluate the protection properties of these combinations as an improved tuberculosis subunit vaccine.     

Experimental Mycoplasma pulmonis Infection in Pathogen-Free Mice: Models for Studying Mycoplasmosis of the Respiratory Tract

Mice of a Swiss substrain, reared under rigid pathogen-free (PF) conditions, were inoculated intranasally with broth cultures of Mycoplasma pulmonis ranging in dose from 10¹ to 9 × 10⁹ colony forming units (CFU). A highly reproducible disease resulted with an LD₅₀ of 1.3 × 10⁸ CFU and a PD₅₀ (dose producing pneumonia in 50% of mice) of 3.4 × 10⁵ CFU. The inoculating dose of M pulmonis was found to be the critical determinant of the severity, duration and pathologic character of the respiratory disease produced. PF mice given 10⁴ CFU or less developed a transient illness characterized by low frequencies of rhinitis, otitis media, laryngotracheitis and focal pneumonia. This was proposed as a low dose model. Doses of 10⁵ to 10⁹ CFU resulted in high frequencies of rhinitis, otitis media, laryngotracheitis and pneumonia. Within the first 10 days the pneumonia often was fatal, being characterized by an outpouring of neutrophils and edema fluid into alveolar spaces, pulmonary congestion and hemorrhage and, occasionally, pleuritis. This high dose—acute disease model was shown to be the result of seeding alveoli with large numbers of organisms at the time of intranasal inoculation. In animals surviving doses of 10⁵ to 10⁹ CFU beyond approximately 10 days postinoculation, the larger concentration of organisms was present in bronchi and bronchioles, giving rise to a third model, the high dose—chronic disease model. The predominant lesions were chronic suppurative bronchitis and bronchiolitis, marked peribronchial lymphoid cuffing, variable numbers of neutrophils and macrophages in alveoli, and complications such as bronchiectasis and pulmonary abscesses. Identical lesions were observed in axenic mice infected with M pulmonis. The infection in PF mice is considered a highly useful experimental system, both for comparative study of respiratory mycoplasmosis and for investigations directed toward understanding and eliminating the natural disease this agent causes in conventional mice and rats.