Mycobacterial codon optimization enhances antigen expression and virus-specific immune responses in recombinant Mycobacterium bovis bacille Calmette-Guérin expressing human immunodeficiency virus type 1 Gag

Although its potential for vaccine development is already known, the introduction of recombinant human immunodeficiency virus (HIV) genes to Mycobacterium bovis bacille Calmette-Guérin (BCG) has thus far elicited only limited responses. In order to improve the expression levels, we optimized the codon usage of the HIV type 1 (HIV-1) p24 antigen gene of gag (p24 gag) and established a codon-optimized recombinant BCG (rBCG)-p24 Gag which expressed a 40-fold-higher level of p24 Gag than did that of nonoptimized rBCG-p24 Gag. Inoculation of mice with the codon-optimized rBCG-p24 Gag elicited effective immunity, as evidenced by virus-specific lymphocyte proliferation, gamma interferon ELISPOT cell induction, and antibody production. In contrast, inoculation of animals with the nonoptimized rBCG-p24 Gag induced only low levels of immune responses. Furthermore, a dose as small as 0.01 mg of the codon-optimized rBCG per animal proved capable of eliciting immune responses, suggesting that even low doses of a codon-optimized rBCG-based vaccine could effectively elicit HIV-1-specific immune responses.     

Cell-mediated immunity induced by recombinant Mycobacterium bovis Bacille Calmette-Guérin strains against an intracellular bacterial pathogen: importance of antigen secretion or membrane-targeted antigen display as lipoprotein for vaccine efficacy

Live recombinant vaccines expressing defined pathogen-derived Ags represent powerful candidates for future vaccination strategies. In this study, we report on the differential induction of protective cell-mediated immunity elicited by different recombinant Mycobacterium bovis Bacille Calmette-Guérin (BCG) strains displaying p60 Ag of Listeria monocytogenes in secreted, cytosolic, or membrane-attached form for T cell recognition. Anti-listerial protection evoked by the membrane-linked p60 lipoprotein of rBCG Mp60 and that of the p60 derivative secreted by rBCG Sp60-40 were nearly equal, whereas cytosolic p60 displayed by rBCG Np60 failed to protect mice from listeriosis. In vivo depletion of CD4 or CD8 T cell subpopulations in rBCG Mp60-vaccinated mice before listerial challenge revealed interactions of both T cell subsets in anti-listerial protection. In rBCG Sp60-40-vaccinated animals, CD4 T cells predominantly contributed to anti-listerial control as shown by the failure of anti-CD8 mAb treatment to impair the outcome of listeriosis in rBCG Sp60-40-vaccinated mice after L. monocytogenes challenge. Hence, differential Ag display by rBCG influences cell-mediated immunity, which in turn may impact vaccine efficacy due to the different requirements of CD4 or CD8 T cells for pathogen elimination.     

Overexpression of IL-15 in vivo enhances protection against Mycobacterium bovis bacillus Calmette-Guérin infection via augmentation of NK and T cytotoxic 1 responses.

To investigate the immunomodulating effects of IL-15 in vivo on mycobacterial infection, we used IL-15-transgenic (Tg) mice, which were recently constructed with cDNA-encoding secretable isoform of IL-15 precursor protein under the control of a MHC class I promoter. The IL-15-Tg mice exhibited resistance against infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG), as assessed by bacteria growth. IFN-gamma level in serum was significantly higher in IL-15-Tg mice than in non-Tg mice after BCG infection. NK cells were remarkably increased, and Ag-specific T cytotoxic 1 response mediated by CD8+ T cells producing IFN-gamma was significantly augmented in the IL-15-Tg mice following BCG infection. Neutralization of endogenous IFN-gamma by in vivo administration of anti-IFN-gamma mAb deteriorated the clearance of the bacteria. Depletion of of NK cells or CD8+ T cells by in vivo administration of anti-asialo-GM(1) Ab or anti-CD8 mAb hampered the exclusion of bacteria. Thus, overexpression of IL-15 in vivo enhanced protection against BCG infection via augmentation of NK and T cytotoxic 1 responses.     

Vaccination of rhesus macaques with recombinant Mycobacterium bovis bacillus Calmette-Guérin Env V3 elicits neutralizing antibody-mediated protection against simian-human immunodeficiency virus with a homologous but not a heterologous V3 motif

Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD). Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific. Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1BZ167/X4, HIV-1SF2/X4, HIV-1CI2/X4, and, to a lesser extent, HIV-1MNp/X4, all of which contain a V3 sequence homologous to that of rBCG Env V3. In contrast, neutralization was not observed against HIV-1SF33/X4, which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B. Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge. In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques. Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV. These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations.     

The secretion antigen SA5K has a role in the adaptation of Mycobacterium bovis bacillus Calmette-Guérin to intracellular stress and hypoxia

The Mycobacterium tuberculosis TB8.4 (Rv1174c) gene encodes a secreted protein of 8.4 kDa (TB8.4) which has been suggested to be involved in reactivation of dormant mycobacteria. We have previously reported that inactivation of an identical gene (sa5k) in Mycobacterium bovis BCG causes impaired ability of the mutant strain (BCGsa5k::aph) to grow inside human macrophages. This study aimed to investigate the role of TB8.4 in the reactivation of aged cultures of BCG as well as the role of the sa5k gene in the resistance of BCG to intracellular stress conditions and adaptation to hypoxia. Although when added to aged cultures of BCG, TB8.4 caused a statistically significant increase in the number of colony-forming units, a similar effect was obtained in cultures incubated with BSA, suggesting a non-specific growth stimulation by TB8.4. Compared to parental BCG, the BCGsa5k::aph strain showed an increased susceptibility to reactive oxygen and nitrogen intermediates and to acid stress and an impaired ability to adapt to reduced O2 concentrations, when tested in the oxygen-limited Wayne culture system. These results suggest that the product of the sa5k gene (SA5K protein) has a role in both resistance of BCG to intracellular stress and in its adaptation to hypoxia.     

Identification of novel proteins in culture filtrates of Mycobacterium bovis bacillus Calmette-Guérin in the isoelectric point range 6-11

Two-dimensional gel electrophoresis and mass spectrometry were used to identify proteins in the isoelectric point range 6-11 in culture filtrates of Mycobacterium bovis bacillus Calmette-Guérin (BCG). Twelve proteins were identified, three of which had not been described previously. The expression of the identified proteins was comparatively analyzed in culture filtrates of BCG in different growth phases and culture conditions. For some of these proteins, the relative protein abundance in the different culture filtrate preparations was significantly different. The differential expression of the identified proteins is discussed in relation to their putative localization and/or biological function.     

Mycobacterium bovis bacillus Calmette-Guérin secreting active cathepsin S stimulates expression of mature MHC class II molecules and antigen presentation in human macrophages

A successful Th cell response to bacterial infections is induced by mature MHC class II molecules presenting specific Ag peptides on the surface of macrophages. In recent studies, we demonstrated that infection with the conventional vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) specifically blocks the surface export of mature class II molecules in human macrophages by a mechanism dependent on inhibition of cathepsin S (Cat S) expression. The present study examined class II expression in macrophages infected with a rBCG strain engineered to express and secrete biologically active human Cat S (rBCG-hcs). Cat S activity was completely restored in cells ingesting rBCG-hcs, which secreted substantial levels of Cat S intracellularly. Thus, infection with rBCG-hcs, but not parental BCG, restored surface expression of mature MHC class II molecules in response to IFN-gamma, presumably as result of MHC class II invariant chain degradation dependent on active Cat S secreted by the bacterium. These events correlated with increased class II-directed presentation of mycobacterial Ag85B to a specific CD4(+) T cell hybridoma by rBCG-hcs-infected macrophages. Consistent with these findings, rBCG-hcs was found to accelerate the fusion of its phagosome with lysosomes, a process that optimizes Ag processing in infected macrophages. These data demonstrated that intracellular restoration of Cat S activity improves the capacity of BCG-infected macrophages to stimulate CD4(+) Th cells. Given that Th cells play a major role in protection against tuberculosis, rBCG-hcs would be a valuable tuberculosis vaccine candidate.     

The shift of Th1 to Th2 immunodominance associated with the chronicity of Mycobacterium bovis bacille Calmette-Guérin infection does not affect the memory response

In the present study we investigated the shaping and evolution of the immunodominance of the T cell response during a chronic mycobacterial infection. Using a recombinant bacille Calmette-Guérin expressing a reporter Ag, the Escherichia coli MalE protein, we analyzed the peptide specificity and the cytokine profile of the T cell response to the reporter Ag by ELISPOT. During the early steps of infection, the T cell response was focused on two dominant MalE epitopes and was characterized by a pure IFN-gamma response. Then, in the course of infection the initial IFN-gamma response to these two epitopes shifted to a mixed IFN-gamma/IL-4 response. At the same time, the peptide specificity of the T cell response was broadened to two additional MalE epitopes characterized by a unique IL-4 response resulting in the establishment of a dominant IL-4 response to the MalE protein at 16 wk postinfection. However, this phenomenon did not impair the outcome of a predominant IFN-gamma response upon subsequent MalE recall in vivo performed in the presence of CFA, a Th1-driving adjuvant. These results indicate that the Th2 nature of the immune response established during a chronic infection, which most likely reflects regulatory mechanisms to allow the return to T cell homeostasis, does not shape the Th1/Th2 nature of the memory response.     

A reduced antigen load in vivo, rather than weak inflammation, causes a substantial delay in CD8+ T cell priming against Mycobacterium bovis (bacillus Calmette-Guérin)

Regardless of the dose of Ag, Ag presentation occurs rapidly within the first few days which results in rapid expansion of the CD8+ T cell response that peaks at day 7. However, we have previously shown that this rapid priming of CD8+ T cells is absent during infection of mice with Mycobacterium bovis (bacillus Calmette-Guérin (BCG)). In this study, we have evaluated the mechanisms responsible for the delayed CD8+ T cell priming. Because BCG replicates poorly and survives within phagosomes we considered whether 1) generation of reduced amounts of Ag or 2) weaker activation by pathogen-associated molecular patterns (PAMPs) during BCG infection is responsible for the delay in CD8+ T cell priming. Using rOVA-expressing bacteria, our results indicate that infection of mice with BCG-OVA generates greatly reduced levels of OVA, which are 70-fold lower in comparison to the levels generated during infection of mice with Listeria monocytogenes-expressing OVA. Furthermore, increasing the dose of OVA, but not PAMP signaling during BCG-OVA infection resulted in rapid Ag presentation and consequent expansion of the CD8+ T cell response, indicating that the generation of reduced Ag levels, not lack of PAMP-associated inflammation, was responsible for delayed priming of CD8+ T cells. There was a strong correlation between the relative timing of Ag presentation and the increase in the level of OVA in vivo. Taken together, these results reveal that some slowly replicating pathogens, such as mycobacteria, may facilitate their chronicity by generating reduced Ag levels which causes a substantial delay in the development of acquired immune responses.     

Transmembrane TNF induces an efficient cell-mediated immunity and resistance to Mycobacterium bovis bacillus Calmette-Guérin infection in the absence of secreted TNF and lymphotoxin-alpha

The contribution of a transmembrane (Tm) form of TNF to protective immunity against Mycobacterium bovis bacillus Calmette-Guérin (BCG) was studied in transgenic (tg) mice expressing a noncleavable Tm TNF but lacking the TNF/lymphotoxin-alpha (LT-alpha) locus (Tm TNF tg mice). These mice were as resistant to BCG infection as wild-type mice, whereas TNF/LT-alpha(-/-), TNF(-/-), and LT-alpha(-/-) mice succumbed. Tm TNF tg mice developed granulomas of smaller size but at 2- to 4-fold increased frequencies compared with wild-type mice. Granulomas were mainly formed by monocytes and activated macrophages expressing Tm TNF mRNA and accumulating acid phosphatase. NO synthase 2 activation as a key macrophage bactericidal mechanism was low during the acute phase of infection in Tm TNF tg mice but was still sufficient to limit bacterial growth and increased in late infection. While infection with virulent Mycobacterium tuberculosis resulted in very rapid death of TNF/LT-alpha(-/-) mice, it also resulted in survival of Tm TNF tg mice which presented an increase in the number of CFU in spleen (5-fold) and lungs (10-fold) as compared with bacterial load of wild-type mice. In conclusion, the Tm form of TNF induces an efficient cell-mediated immunity and total resistance against BCG even in the absence of LT-alpha and secreted TNF. However, Tm TNF-mediated protection against virulent M. tuberculosis infection can also be efficient but not as strong as in BCG infection, in which cognate cellular interactions may play a more predominant role in providing long-term surveillance and containment of BCG-infected macrophages.     

Importance of L3T4+ and Lyt-2+ cells in the immunologic control of infection with Mycobacterium bovis strain bacillus Calmette-Guérin in mice. Assessment by elimination of T cell subsets in vivo

The course of infection after injection of small doses of bacillus Calmette-Guérin (BCG) was studied in mice which were depleted in vivo of T cell subsets by administration of either anti-L3T4 or anti-Lyt-2 mAb. The results presented herein strongly suggest that the L3T4+ subpopulation play a pivotal role in the immunologic control of BCG infection because the depletion of L3T4+ cells led to a dramatic increase in the number of viable bacteria. Depletion of Lyt-2+ cells had no significant effect on the course of infection. These results were confirmed by using adoptive transfer experiments which showed that protective immunity was mediated by L3T4+ cells generated in the spleen as a result of infection. Moreover, T cells capable of controlling the recurrence of BCG multiplication from residual bacteria remaining in organs after the recovery from infection were shown to belong to the L3T4+ subpopulation.     

Mycobacterium bovis bacillus Calmette-Guérin (BCG)-induced CXCL8 production is mediated through PKCalpha-dependent activation of the IKKalphabeta signaling pathway in epithelial cells

Upon contact with airway epithelial cells, mycobacteria activate several signal transduction events that are required for induction of NF-kappaB-dependent chemokine gene expression. However, downstream signaling pathways, especially that of Ca(2+)-dependent protein kinase C alpha (PKCalpha), and in particular, the identity of the IKKalphabeta signal pathway for CXCL8 secretion in Mycobacterium bovis BCG-induced epithelial cells are still unknown. In this study, we demonstrated that the phosphoinositide-phospholipase C (PI-PLC) downstream signaling pathway is involved in M. bovis BCG-induced CXCL8 release, since A549 cells pretreated with U73122, a PI-PLC inhibitor, inhibited CXCL8 release, whereas U73343 the inactive analog had no effect. In addition, our results demonstrated that M. bovis BCG-induced CXCL8 production by A549 cells was significantly blocked by using neomycin (another well-described inhibitor of PI-PLC with a different mechanism of action), Ro-32-0432 and Ro-31-8220 (two PKCalpha inhibitors), PP1 and PP2 (two potent and selective inhibitors of the Src-family tyrosine kinases), and Bay 11-7082 (an IkappaB phosphorylation inhibitor). We also demonstrated that M. bovis BCG can rapidly induce translocation of PKCalpha from the cytosol to the membrane, and that treatment of cells with M. bovis BCG caused time-dependent increases in phosphorylation of c-Src at tyrosine 416. Finally, our studies revealed that M. bovis BCG induced the association of c-Src and IKKalphabeta during the interaction of PKCalpha and IKKalphabeta. Altogether, these results represent the first evidence to date suggesting that M. bovis BCG activates the PI-PLC/PKCalpha/c-Src/IKKalphabeta signaling pathway to induce CXCL8 release in human epithelial cells.     

Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.     

A nonfucosylated variant of the anti-HIV-1 monoclonal antibody b12 has enhanced FcγRIIIa-mediated antiviral activity in vitro but does not improve protection against mucosal SHIV challenge in macaques

Eliciting neutralizing antibodies is thought to be a key activity of a vaccine against human immunodeficiency virus (HIV). However, a number of studies have suggested that in addition to neutralization, interaction of IgG with Fc gamma receptors (FcγR) may play an important role in antibody-mediated protection. We have previously obtained evidence that the protective activity of the broadly neutralizing human IgG1 anti-HIV monoclonal antibody (MAb) b12 in macaques is diminished in the absence of FcγR binding capacity. To investigate antibody-dependent cellular cytotoxicity (ADCC) as a contributor to FcγR-associated protection, we developed a nonfucosylated variant of b12 (NFb12). We showed that, compared to fully fucosylated (referred to as wild-type in the text) b12, NFb12 had higher affinity for human and rhesus macaque FcγRIIIa and was more efficient in inhibiting viral replication and more effective in killing HIV-infected cells in an ADCC assay. Despite these more potent in vitro antiviral activities, NFb12 did not enhance protection in vivo against repeated low-dose vaginal challenge in the simian-human immunodeficiency virus (SHIV)/macaque model compared to wild-type b12. No difference in protection, viral load, or infection susceptibility was observed between animals given NFb12 and those given fully fucosylated b12, indicating that FcγR-mediated activities distinct from FcγRIIIa-mediated ADCC may be important in the observed protection against SHIV challenge.