Effects of antenatal and postnatal environments on CD4 T-cell responses to Mycobacterium bovis BCG in healthy infants in the Gambia

The Mycobacterium bovis BCG vaccine has a poor record of efficacy in low-income tropical settings. Against this background, we evaluated the immune response of infants to mycobacterial antigens over the 2 years following BCG vaccination at birth by measuring the gamma interferon (IFN-gamma), interleukin-2 (IL-2), and CD154 responses of CD4 T cells. Similar numbers of cells expressed IFN-gamma in infants, 4- to 5-year-old children, and adults, while CD154 was not expressed at comparable levels until the second year of infancy. The IL-2 response remained relatively low in infants, children, and adults but correlated negatively with mother's body mass index and was highest among infants born to Mandinka mothers. Similarly, infants born in the wet season had a stronger CD154 response than those born in the dry season throughout the 2 years of the study. We conclude that the prenatal and perinatal environments have a lasting effect on the response of infants to the BCG vaccine.     

The influence of previous exposure to environmental mycobacteria on the interferon-gamma response to bacille Calmette-Guérin vaccination in southern England and northern Malawi

We report a large study of the effect of BCG vaccination on the in vitro 6-day whole blood interferon-gamma (IFN-gamma) response to antigens from eight species of mycobacteria among schoolchildren in south-eastern England, where bacille Calmette-Guérin (BCG) vaccination is highly protective against pulmonary tuberculosis, and among young adults in northern Malawi, where BCG vaccination is not protective. In the UK children, BCG induced an appreciable increase in IFN-gamma response to antigens from most species of mycobacteria. The degree of change was linked to the relatedness of the species to Mycobacterium bovis BCG, and provides further evidence of the cross-reactivity of mycobacterial species in priming of the immune system. IFN-gamma responses to purified protein derivatives (PPDs) from M. tuberculosis and environmental mycobacteria were more prevalent in the Malawian than the UK group prior to vaccination; BCG vaccination increased the prevalence of responses to these PPDs in the UK group to a level similar to that in Malawi. There was no evidence that the vaccine-induced change in IFN-gamma response was dependent upon the magnitude of the initial response of the individual to environmental mycobacteria in the United Kingdom or in Malawi. These observations should assist the development and interpretation of human clinical trials of new vaccines against M. tuberculosis in areas of both low and high exposure to environmental mycobacteria.     

Optimizing HIV-1-specific CD8+ T-cell induction by recombinant BCG in prime-boost regimens with heterologous viral vectors

The desire to induce HIV-1-specific responses soon after birth to prevent breast milk transmission of HIV-1 led us to propose a vaccine regimen which primes HIV-1-specific T cells using a recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) vaccine. Because attenuated live bacterial vaccines are typically not sufficiently immunogenic as stand-alone vaccines, rBCG-primed T cells will likely require boost immunization(s). Here, we compared modified Danish (AERAS-401) and Pasteur lysine auxotroph (222) strains of BCG expressing the immunogen HIVA for their potency to prime HIV-1-specific responses in adult BALB/c mice and examined four heterologous boosting HIVA vaccines for their immunogenic synergy. We found that both BCG.HIVA(401) and BCG.HIVA(222) primed HIV-1-specific CD8(+) T-cell-mediated responses. The strongest boosts were delivered by human adenovirus-vectored HAdV5.HIVA and sheep atadenovirus-vectored OAdV7.HIVA vaccines, followed by poxvirus MVA.HIVA; the weakest was plasmid pTH.HIVA DNA. The prime-boost regimens induced T cells capable of efficient in vivo killing of sensitized target cells. We also observed that the BCG.HIVA(401) and BCG.HIVA(222) vaccines have broadly similar immunologic properties, but display a number of differences mainly detected through distinct profiles of soluble intercellular signaling molecules produced by immune splenocytes in response to both HIV-1- and BCG-specific stimuli. These results encourage further development of the rBCG prime-boost regimen.     

Gamma interferon responses induced by a panel of recombinant and purified mycobacterial antigens in healthy,non-mycobacterium bovis BCG-vaccinated Malawian young adults

We have previously shown that young adults living in a rural area of northern Malawi showed greater gamma interferon (IFN-gamma) responses to purified protein derivatives (PPD) prepared from environmental mycobacteria than to PPD from Mycobacterium tuberculosis. In order to define the mycobacterial species to which individuals living in a rural African population have been exposed and sensitized, we tested T-cell recognition of recombinant and purified antigens from M. tuberculosis (38 kDa, MPT64, and ESAT-6), M. bovis (MPB70), M. bovis BCG (Ag85), and M. leprae (65 kDa, 35 kDa, and 18 kDa) in >600 non-M. bovis BCG-vaccinated young adults in the Karonga District of northern Malawi. IFN-gamma was measured by enzyme-linked immunosorbent assay (ELISA) in day 6 supernatants of diluted whole-blood cultures. The recombinant M. leprae 35-kDa and 18-kDa and purified native M. bovis BCG Ag85 antigens induced the highest percentages of responders, though both leprosy and bovine tuberculosis are now rare in this population. The M. tuberculosis antigens ESAT-6 and MPT64 and the M. bovis antigen MPB70 induced the lowest percentages of responders. One of the subjects subsequently developed extrapulmonary tuberculosis; this individual had a 15-mm-diameter reaction to the Mantoux test and responded to M. tuberculosis PPD, Ag85, MPT64, and ESAT-6 but not to any of the leprosy antigens. We conclude that in this rural African population, exposure to M. tuberculosis or M. bovis is much less frequent than exposure to environmental mycobacteria such as M. avium, which have antigens homologous to the M. leprae 35-kDa and 18-kDa antigens. M. tuberculosis ESAT-6 showed the strongest association with the size of the Mantoux skin test induration, suggesting that among the three M. tuberculosis antigens tested it provided the best indication of exposure to, or infection with, M. tuberculosis.     

PCR identification of Mycobacterium bovis BCG.

The attenuated bacillus Calmette-Guérin (BCG) vaccine strain is derived from a virulent strain of Mycobacterium bovis. BCG is difficult to differentiate from other strains of M. bovis and other members of the M. tuberculosis complex by conventional methods. Recently, a genomic region designated RD1 was found to be present in all virulent M. bovis and M. tuberculosis strains tested but deleted from all BCG strains tested. With this information, a multiplex PCR method was developed to detect the RD1 deletion. A large collection of BCG and other M. tuberculosis complex strains from diverse host and geographic origins was tested. RD1 was deleted in 23 of 23 BCG strains. RD1 was present in 129 of 129 other M. tuberculosis complex strains. This multiplex PCR method can be used as a tool for the rapid and specific identification of BCG.     

A DNA prime-live vaccine boost strategy in mice can augment IFN-gamma responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis

The Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine has variable efficacy for both human and bovine tuberculosis. There is a need for improved vaccines or vaccine strategies for control of these diseases. A recently developed prime-boost strategy was investigated for vaccination against M. bovis infection in mice. BALB/c and C57BL/6 mice were primed with a DNA vaccine, expressing two mycobacterial antigens, ESAT-6 and antigen 85 A and boosted with attenuated M. bovis strains, BCG or WAg520, a newly attenuated strain, prior to aerosol challenge. Before challenge, the antigen-specific production of interferon-gamma (IFN-gamma) was evaluated by ELISPOT and antibody responses were measured. The prime-boost stimulated an increase in the numbers of IFN-gamma producing cells compared with DNA or live vaccination alone, but this varied according to the attenuated vaccine strain, time of challenge and the strain of mouse used. Animals vaccinated with DNA alone generated the strongest antibody response to mycobacterial antigens, which was predominantly IgG1. BCG and WAg520 alone generally gave a 1-2 log10 reduction in bacterial load in lungs or spleen, compared to non-vaccinated or plasmid DNA only control groups. The prime-boost regimen was not more effective than BCG or WAg520 alone. These observations demonstrate the comparable efficacy of BCG and WAg520 in a mouse model of bovine tuberculosis. However, priming with the DNA vaccine and boosting with an attenuated M. bovis vaccine enhanced IFN-gamma immune responses compared to vaccinating with an attenuated M. bovis vaccine alone, but did not increase protection against a virulent M. bovis infection.     

Immunoassays of field isolates of Mycobacterium bovis and other mycobacteria by use of monoclonal antibodies

Antigen extracts obtained by sonication of 22 strains of Mycobacterium bovis from cattle and badgers together with extracts of strains of M. tuberculosis, M. paratuberculosis, M. avium, M. africanum, M. kansasi, M. leprae and BCG were examined with a panel of 10 monoclonal antibodies to M. tuberculosis or M. leprae. Antigen extracts were coated in aqueous solution (wet coating) and the extracts were also dried on to the polyvinyl plates (dry coating). When dry coating was compared to wet coating, there was a major increase in the binding of monoclonal antibody ML03 to M. avium and M. paratuberculosis, monoclonal antibody ML02 to M. paratuberculosis, and monoclonal antibodies TB71 and TB72 to the majority of M. bovis isolates. The study confirmed that on wet-coated plates, monoclonal antibodies TB71 and TB72 bind poorly or not at all to M. bovis and that monoclonal antibodies TB68, TB78, TB77 and TB23 each bind to field strains of M. bovis while TB23 binds poorly to BCG in wet-coating conditions. Antibodies TB72 and TB71, originally thought to be specific for M. tuberculosis, each reacted with M. africanum. Antibody TB78 bound to M. paratuberculosis but did not react with M. avium, and M. avium and M. paratuberculosis were distinguished from M. bovis and M. tuberculosis by the binding of antibody ML03 to dry-coated plates. When wet-coated plates were used, ML03 bound strongly only to M. leprae. The panel of monoclonal antibodies did not demonstrate distinct serotype differences between the field isolates of M. bovis.     

Inhibition of TNF-α-induced Cyclooxygenase-2 Expression by Mycobacterium bovis BCG in Human Alveolar Epithelial A549 Cells

Cyclooxygenase-2 (COX-2) is implicated in pathophysiological processes associated with the initiation or maintenance of host inflammatory responses to infection. Our results demonstrates that Mycobacterium bovis BCG ( M. bovis BCG) downregulates tumour necrosis factor-α (TNF-α)-induced COX-2 gene expression in alveolar epithelial cells by inhibiting the phosphorylations of Raf-1 and p38 kinases. Further, M. bovis BCG-mediated inhibition of COX-2 or p38 mitogen-activated protein kinase could be reversed by Calyculin A, a selective inhibitor of Ser/Thr phosphatases. Moreover, M. bovis BCG inhibited the TNF-α-triggered NF-κB activation following IκB degradation. Taken together, these results suggest that the attenuation of COX-2 expression by vaccine strain, M. bovis BCG, represents a novel strategy to maintain robust host proinflammatory responses to subsequent challenges with virulent tuberculosis bacilli.     

Identification of surrogates and correlates of protection in protective immunity against Mycobacterium bovis infection induced in neonatal calves by vaccination with M. bovis BCG Pasteur and M. bovis BCG Danish

Vaccination of neonatal calves with Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces a significant degree of protection against infection with virulent M. bovis, the causative agent of bovine tuberculosis (bTB). We compared two strains of BCG, Pasteur and Danish, in order to confirm that the current European human vaccine strain (BCG Danish) induced protective immunity in calves, and we assessed immune responses to determine correlates of protection that could assist future vaccine evaluation in cattle. Both vaccine strains induced antigen (purified protein derivate [PPD])-specific gamma interferon (IFN-γ) in whole-blood cultures. These responses were not significantly different for BCG Pasteur and BCG Danish and peaked at week 2 to 4 postvaccination. Vaccination with either BCG Danish or BCG Pasteur induced significant protection against bTB, with reductions in both lesion score and bacteriological burden evident in both groups of vaccinated calves compared with nonvaccinated control calves. Measurement of IFN-γ-expressing T lymphocytes postvaccination and postchallenge revealed both correlates and surrogates of protective efficacy. The frequency of central memory T lymphocytes present at 12 weeks postvaccination (at the time of M. bovis challenge) correlated significantly with protection. Conversely, the number of IFN-γ-expressing effector T cells present after M. bovis challenge was correlated with disease. These results demonstrate that vaccination of neonatal calves with either BCG Pasteur or BCG Danish induces protective immune responses against TB. In addition, we show that measurement of antigen-specific T lymphocyte populations may provide a reliable means for identifying protective vaccine candidates.     

Immunogenicity and protection studies with recombinant mycobacteria and vaccinia vectors coexpressing the 18-kilodalton protein of Mycobacterium leprae.

The activation of antigen-specific T lymphocytes is essential for the control of leprosy infection in humans and experimental animals. T cells recognize a variety of protein antigens from Mycobacterium leprae, including the 18-kDa protein, which is limited in distribution among mycobacteria and which is absent from Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG. Adjuvant preparations of mycobacterial protein antigens have had limited protective efficacy for experimental infections in animals. Since recombinant vectors may elicit more effective T-cell responses than adjuvant preparations, recombinant vaccinia virus (VV18) and M. bovis BCG (BCG18) vectors expressing the 18-kDa protein of M. leprae were prepared. Both VV18 and BCG18 stimulated anti-18-kDa protein antibody and lymphocyte proliferative responses. Sequential immunization with VV18 followed by BCG18 induced higher levels of specific immunoglobulin G2a antibodies than immunoglobulin G1 antibodies, in contrast to immunization with VV18 or BCG18 alone. The protective efficacy of immunization with VV18 from a challenge with BCG18 was examined in two murine models of mycobacterial infection. After intravenous challenge, mice immunized with recombinant vaccinia virus exhibited lower initial levels of replication and earlier clearance of BCG18 from their spleens than mice immunized with vaccinia virus expressing an unrelated protein. After footpad infection in a dissemination model, there was earlier clearance of BCG18 from specifically immunized mice. However, immunization of mice with VV18 did not prevent a productive mycobacterial infection.     

Microbicidal activity and morphological characteristics of lung macrophages in Mycobacterium bovis BCG cell wall-induced lung granuloma in mice.

Morphological and functional changes in lung macrophages from mice injected intravenously with Mycobacterium bovis BCG cell walls (CWs) were studied. In BCG CW high-responder mice (C57BL/6 [B6] strain), an increase in the size and the acid phosphatase activity of lung macrophages was observed. These lung macrophages showed greater microbicidal activity to M. bovis Ravenel and Listeria monocytogenes EGD, enhanced superoxide anion production index, and greater macrophage migration inhibition activity, as compared with lung macrophages from BCG CW low-responder mice (C3H/He strain), which were small in size and showed weak acid phosphatase activity, low antimicrobial activity, and low superoxide anion production index upon intravenous injection of the mice with BCG CW. These results indicated that lung macrophages from B6 mice injected with BCG CWs were morphologically and functionally activated, but not those from C3H mice.     

Cloning of a species-specific antigen of Mycobacterium bovis.

A DNA library from a virulent strain of Mycobacterium bovis was constructed in the expression vector lambda gt11, and the library was probed with antisera to M. bovis. Clones expressing M. bovis antigens were isolated and characterized by using M. bovis-specific monoclonal antibodies that recognize a 22,000-molecular-weight protein (MPB70). MPB70 is a major protein antigen of the vaccine strain of M. bovis BCG and of virulent M. bovis, the causative agent of bovine tuberculosis. Of 32 clones selected by using polyclonal affinity-purified anti-M. bovis sera, 5 were recognized by the anti-MPB-70 monoclonal antibodies, and one monoclonal antibody, SB10, recognized all 5 clones. Characterization of these clones showed that one clone containing a 253-base-pair insert expressed a polypeptide bound by all of the MPB70-specific monoclonal antibodies. Western blots (immunoblots) showed that this cloned protein was recognized by sera from M. bovis-infected cattle, although not all cattle with bovine tuberculosis produced antibodies reactive to this clone. DNA sequencing of the clone showed that it coded for 84 amino acids from positions 17 to 114 of the 161-amino-acid protein, with a 16-peptide deletion between positions 79 and 94. Apart from this deletion, there were seven other variations between the cloned sequence and that deduced from M. bovis BCG MPB70.     

Mycobacterial purified protein derivatives stimulate innate immunity: Malawians show enhanced tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-10 responses compared to those of adolescents in the United Kingdom

To investigate the role of innate immunity in variable efficacy of Mycobacterium bovis BCG vaccination in Malawi and the United Kingdom, we examined 24-h tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-10 responses to mycobacterial purified protein derivatives (PPDs). The rank order in stimulatory potency for different PPDs was the same for all three cytokines. Before vaccination Malawians made higher pro- and anti-inflammatory responses than did United Kingdom subjects. Fewer than 5% of United Kingdom subjects made IL-10 in response to any PPD, compared to 19 to 57% responders among Malawians. Priming for regulatory IL-10 may contribute to the smaller increase in gamma interferon responses in Malawians compared to United Kingdom subjects following BCG vaccination.     

The Mycobacterium bovis BCG cyclic AMP receptor-like protein is a functional DNA binding protein in vitro and in vivo, but its activity differs from that of its M. tuberculosis ortholog, Rv3676

Mycobacterium tuberculosis Rv3676 encodes a cyclic AMP (cAMP) receptor-like protein (CRP(Mt)) that has been implicated in global gene regulation and may play an important role during tuberculosis infection. The CRP(Mt) ortholog in Mycobacterium bovis BCG, CRP(BCG), is dysfunctional in an Escherichia coli CRP competition assay and has been proposed as a potential source of M. bovis BCG's attenuation. We compared CRP(BCG) and CRP(Mt) in vitro and in vivo, in M. bovis BCG and M. tuberculosis, to evaluate CRP(BCG)'s potential function in a mycobacterial system. Both proteins formed dimers in mycobacterial lysates, bound to the same target DNA sequences, and were similarly affected by the presence of cAMP in DNA binding assays. However, CRP(Mt) and CRP(BCG) differed in their relative affinities for specific DNA target sequences and in their susceptibilities to protease digestion. Surprisingly, CRP(BCG) DNA binding activity was stronger than that of CRP(Mt) both in vitro and in vivo, as measured by electrophoretic mobility shift and chromatin immunoprecipitation assays. Nutrient starvation-associated regulation of several CRP(Mt) regulon members also differed between M. bovis BCG and M. tuberculosis. We conclude that CRP(BCG) is a functional cAMP-responsive DNA binding protein with an in vivo DNA binding profile in M. bovis BCG similar to that of CRP(Mt) in M. tuberculosis. However, biologically significant functional differences may exist between CRP(BCG) and CRP(Mt) with respect to gene regulation, and this issue warrants further study.     

A case of sepsis caused by BCG (Bacillus Calmette Guerin) after its bladder instillation in a 71-year-old patient affected by bladder carcinoma

Sometimes the Calmette-Guerin bacillus can be used with success as local immunotherapy for superficial bladder carcinoma. BCG is a living attenuated strain of Mycobacterium bovis and therefore its biadder instillation may give rise, in some particular cases, to serious systemic effects, caused either by hypersensitivity reaction or by systemic dissemination in case with low efficiency of cellular immunity. We describe a case of sepsis in a 71-year-old patient after bladder instillation of BCG with a rare complication of granulomatous hepatitis. The etiological diagnosis of granulomatous hepatitis with PCR on liver biopsy is very important for the specific therapy before the patient can undergo a steroid regime.     

Analysis of the relationships between environmental factors (aeroallergens, air pollution, and weather) and asthma emergency admissions to a hospital in Mexico City

There have been several studies of the relationships between environmental factors, particularly air pollution, and attacks of asthma. Most of these studies have ignored the potential confounding effects of aeroallergens such as pollens and fungal spores. We report a statistical analysis of the relationships between emergency admissions for asthma to a hospital in Mexico City and daily average airborne concentrations of pollen, fungal spores, air pollutants (O₃, NO₃, SO₃. and particulates) and weather factors. Asthma admissions had a seasonal pattern with more during the wet season (May-October) than the dry season (November-April). There were few statistical associations between asthma admissions and air pollutants for the three age groups studied (children under 15 years, adults, and seniors [adults over 59 years]) in either season. Grass pollen was associated with child and adult admissions for both the wet and dry seasons, and fungal spores were associated with child admissions during both the wet and dry seasons. The analysis was done with environmental data averaged over the day of admission and the 2 previous days. Our results suggest that aeroallergens may be statistically associated more strongly with asthma hospital admissions than air pollutants and may act as confounding factors in epidemiologic studies.     

A recombinant attenuated Mycobacterium tuberculosis vaccine strain is safe in immunosuppressed simian immunodeficiency virus-infected infant macaques

Many resource-poor countries are faced with concurrent epidemics of AIDS and tuberculosis (TB) caused by human immunodeficiency virus (HIV) and Mycobacterium tuberculosis, respectively. Dual infections with HIV and M. tuberculosis are especially severe in infants. There is, however, no effective HIV vaccine, and the only licensed TB vaccine, the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine, can cause disseminated mycobacterial disease in HIV-infected children. Thus, a pediatric vaccine to prevent HIV and M. tuberculosis infections is urgently needed. We hypothesized that a highly attenuated M. tuberculosis strain containing HIV antigens could be safely administered at birth and induce mucosal and systemic immune responses to protect against HIV and TB infections, and we rationalized that vaccine safety could be most rigorously assessed in immunocompromised hosts. Of three vaccine candidates tested, the recombinant attenuated M. tuberculosis strain mc(2)6435 carrying a simian immunodeficiency virus (SIV) Gag expression plasmid and harboring attenuations of genes critical for replication (panCD and leuCD) and immune evasion (secA2), was found to be safe for oral or intradermal administration to non-SIV-infected and SIV-infected infant macaques. Safety was defined as the absence of clinical symptoms, a lack of histopathological changes indicative of M. tuberculosis infection, and a lack of mycobacterial dissemination. These data represent an important step in the development of novel TB vaccines and suggest that a combination recombinant attenuated M. tuberculosis-HIV vaccine could be a safe alternative to BCG for the pediatric population as a whole and, more importantly, for the extreme at-risk group of HIV-infected infants.     

Dependence of Mycobacterium bovis BCG on Anaerobic Nitrate Reductase for Persistence Is Tissue Specific

Mycobacterium bovis BCG, the only presently available vaccine against tuberculosis, was obtained from virulent M. bovis after serial passages in vitro. The vaccine strain retained at least some of its original virulence, as it persists in immune-competent hosts and occasionally may cause fatal disease in immune-deficient hosts. Mycobacterial persistence in vivo is thought to depend on anaerobic metabolism, an apparent paradox since all mycobacteria are obligate aerobes. Here we report that M. bovis BCG lacking anaerobic nitrate reductase (NarGHJI), an enzyme essential for nitrate respiration, failed to persist in the lungs, liver, and kidneys of immune-competent (BALB/c) mice. In immune-deficient (SCID) mice, however, bacilli caused chronic infection despite disruption of narG, even if growth of the mutant was severely impaired in lungs, liver, and kidneys. Persistence and growth of BCG in the spleens of either mouse strain appeared largely unaffected by lack of anaerobic nitrate reductase, indicating that the role of the enzyme in pathogenesis is tissue specific. These data suggest first that anaerobic nitrate reduction is essential for metabolism of M. bovis BCG in immune-competent but not immune-deficient mice and second that its role in mycobacterial disease is tissue specific, both of which are observations with important implications for pathogenesis of mycobacteria and vaccine development.     

Prophylactic effect of mycobacterium bovis BCG vaccination against osteomyelitis in children with Mycobacterium ulcerans disease (Buruli Ulcer)

Mycobacterium ulcerans disease, or Buruli ulcer (BU), causes significant morbidity in West Africa. In 233 consecutive, laboratory-confirmed samples from BU patients in Benin whose Mycobacterium bovis BCG scar status was known, 130 children (<15 years old) and 75 adults had a neonatal BCG vaccination scar. Of 130 children with BCG scars, 10 (7.7%) had osteomyelitis, while 3 of 9 children without BCG scars (33.3%) had osteomyelitis. Our observations support the conclusion that having a BCG vaccination scar provides significant protection against M. ulcerans osteomyelitis in children with BU disease.     

Secretion of functional monocyte chemotactic protein 3 by recombinant Mycobacterium bovis BCG attenuates vaccine virulence and maintains protective efficacy against M. tuberculosis infection

A strain of Mycobacterium bovis BCG that secretes high levels of functional murine monocyte chemotactic protein 3 (BCG(MCP-3)) was developed. Mice vaccinated with BCG(MCP-3) displayed increased lymphocyte migration in vivo and augmented antigen-specific T-cell responses compared to mice vaccinated with BCG alone. The level of protection afforded by BCG(MCP-3) was equivalent to that with control BCG; however, immunodeficient mice infected with BCG(MCP-3) survived significantly longer than mice infected with the control BCG strain. Therefore, BCG(MCP-3) may be a safer alternative than conventional BCG for vaccination of immunocompromised individuals.