Leander Grode

Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guérin mutants that secrete listeriolysin

The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membrane-perforating listeriolysin (Hly) of Listeria monocytogenes, which was shown to improve protection against Mycobacterium tuberculosis. Following aerosol challenge, the Hly-secreting recombinant BCG (hly+ rBCG) vaccine was shown to protect significantly better against aerosol infection with M. tuberculosis than did the parental BCG strain. The isogenic, urease C-deficient hly+ rBCG (DeltaureC hly+ rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. DeltaureC hly+ rBCG also induced profound protection against a member of the M. tuberculosis Beijing/W genotype family while parental BCG failed to do so consistently. Hly not only promoted antigen translocation into the cytoplasm but also apoptosis of infected macrophages. We concluded that superior vaccine efficacy of DeltaureC hly+ rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell-mediated immunity.

Unique Transcriptome Signature of Mycobacterium tuberculosis in Pulmonary Tuberculosis

ABSTRACT Although tuberculosis remains a substantial global threat, the mechanisms that enable mycobacterial persistence and replication within the human host are ill defined. This study represents the first genome-wide expression analysis of Mycobacterium tuberculosis from clinical lung samples, which has enabled the identification of M. tuberculosis genes actively expressed during pulmonary tuberculosis. To obtain optimal information from our DNA array analyses, we analyzed the differentially expressed genes within the context of computationally inferred protein networks. Protein networks were constructed using functional linkages established by the Rosetta stone, phylogenetic profile, conserved gene neighbor, and operon computational methods. This combined approach revealed that during pulmonary tuberculosis, M. tuberculosis actively transcribes a number of genes involved in active fortification and evasion from host defense systems. These genes may provide targets for novel intervention strategies.

Macrophages of the Splenic Marginal Zone Are Essential for Trapping of Blood-Borne Particulate Antigen but Dispensable for Induction of Specific T Cell Responses

Rapid removal of pathogens from the circulation by secondary lymphoid organs is prerequisite for successful control of infection. Blood-borne Ags are trapped mainly in the splenic marginal zone. To identify the cell populations responsible for Ag trapping in the marginal zone, mice were selectively depleted of marginal zone macrophages and marginal metallophilic macrophages. In the absence of these cells, trapping of microspheres and Listeria monocytogenes organisms was lost, and early control of infection was impaired. Depletion of marginal zone macrophages and marginal metallophilic macrophages, however, did not limit Ag presentation because Listeria-specific protective T cell immunity was induced. Therefore, marginal zone macrophages and marginal metallophilic macrophages are crucial for trapping of particulate Ag but dispensable for Ag presentation.

Recombinant BCG ΔureC hly+ Induces Superior Protection Over Parental BCG by Stimulating a Balanced Combination of Type 1 and Type 17 Cytokine Responses

Background. New vaccines against tuberculosis (TB) are urgently needed because the only available vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), fails to protect against pulmonary TB in adults. The recombinant ΔureC hly+ BCG (rBCG) is more efficient than parental BCG (pBCG) against pulmonary TB in preclinical studies and has proven safe and immunogenic in phase I clinical trials. Methods. In an attempt to identify the mechanisms underlying the superior protection of rBCG, we compared the immune responses elicited after vaccination and subsequent aerosol infection with Mycobacterium tuberculosis (MTB) in mice. Results. We demonstrate that both rBCG and pBCG induce marked type 1 cytokine responses, whereas only rBCG elicits a profound type 17 cytokine response in addition. We observed earlier recruitment of antigen-specific T lymphocytes to the lung upon MTB infection of rBCG-vaccinated mice. These T cells produced abundant type 1 cytokines after restimulation, resulting in 10-fold reduced bacterial burden 90 days after infection. Conclusions. Our findings identify a general immunologic pathway for improved vaccination strategies against TB that can also be harnessed by other vaccine candidates.

Safety and immunogenicity of the recombinant BCG vaccine VPM1002 in a phase 1 open-label randomized clinical trial.

BACKGROUND Current vaccination using Mycobacterium bovis bacillus Calmette-Guérin (BCG), fails to prevent pulmonary tuberculosis (TB). New vaccination strategies are essential for reducing the global incidence of TB. We assessed the safety and immunogenicity of VPM1002, a recombinant BCG vaccine candidate. EudraCT (2007-002789-37) and ClinicalTrials.gov (NCT00749034). METHODS Healthy volunteers were enrolled in a phase 1 open-label, dose escalation randomized clinical trial, and received one intradermal dose of VPM1002 (Mycobacterium bovis BCG ΔureC::hly Hm(R)) or BCG. Immunogenicity was assessed by interferon-gamma (IFN-γ) production, cellular immune response markers by flow cytometry and serum antibodies against mycobacterial antigens. RESULTS Eighty volunteers were randomized into two groups according to previous BCG vaccination and mycobacterial exposure (BCG-naïve, n=40 and BCG-immune, n=40). In each group, 30 individuals were vaccinated with VPM1002 (randomized to three escalating doses) and 10 with BCG. VPM1002 was safe and stimulated IFN-γ-producing and multifunctional T cells, as well as antibody-producing B cells in BCG-naïve and BCG-immune individuals. CONCLUSIONS VPM1002 was safe and immunogenic for B-cell and T-cell responses and hence will be brought forward through the clinical trial pipeline.

Macrophage migration inhibitory factor (MIF) plays a pivotal role in immunity against Salmonella typhimurium

The cytokine macrophage migration inhibitory factor (MIF) exerts a multitude of biological functions. Notably, it induces inflammation at the interface between the immune system and the hypothalamus–pituitary–adrenal stress axis. The role of MIF in infectious diseases is not understood completely. Here, we show that MIF-deficient (MIF−/−) knockout mice fail to control an infection with wild-type Salmonella typhimurium. Increased susceptibility was accompanied by a reduced Th1 response, demonstrated by decreased levels of IL-12, IFNγ, and tumor necrosis factor α. In Salmonella-infected MIF−/− mice, levels of IL-1β were markedly increased. Additionally, infected MIF−/− mice showed elevated serum levels of nitric oxide and corticosterone as compared with control mice. Our results point to MIF as a key mediator in the host response to S. typhimurium. MIF not only promotes development of a protective Th1 response but ameliorates disease by altering levels of reactive nitrogen intermediates and corticosteroid hormones, which both exert immunosuppressive functions.

Application of Mycobacterial Proteomics to Vaccine Design: Improved Protection by Mycobacterium bovis BCG Prime-Rv3407 DNA Boost Vaccination against Tuberculosis

ABSTRACT Information from comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin (BCG) principally allows prediction of potential vaccine candidates. Thirty-six M. tuberculosis DNA vaccine candidates identified by comparative proteome analysis were evaluated in the mouse model for protection against low-dose aerosol M. tuberculosis infection. We identified the DNA vaccine candidate Rv3407 as a protective antigen and analyzed putative major histocompatibility complex class I epitopes by computational predictions and gamma interferon Elispot assays. Importantly, we discovered that the DNA vaccine Rv3407 improved the efficacy of BCG vaccination in a heterologous prime-boost vaccination protocol. Our data demonstrate the rationale of a combination of proteomics, epitope prediction, and broad screening of putative antigens for identification of novel DNA vaccine candidates. Furthermore, our experiments show that heterologous prime-boost vaccination with a defined antigen boost “on top” of a BCG primer provides superior protection against tuberculosis over vaccination with BCG alone.

Recombinant attenuated bacteria for the delivery of subunit vaccines

Using attenuated intracellular bacteria as carriers, we have developed two different approaches for the delivery of subunit vaccines encoding heterologous antigens. The first system is based on the direct secretion of the heterologous antigens in Gram-negative bacteria via the hemolysin secretion system of Escherichia coli into either phagosome or cytosol of infected cells. The second approach is based on the transport of eukaryotic antigen expression vectors by intracellular bacteria like Listeria and Salmonella into the host cell and here, preferably, into the cytosolic compartment. After release of the plasmid DNA from the bacteria, the plasmid-encoded antigens can be expressed directly by the host cell. Finally, we combined both types of subunit vaccines in one live vector - we equipped Salmonella strains with a phagosomal escape function by utilization of the hemolysin secretion system and used this recombinant vaccine strain for the delivery of a eukaryotic antigen expression vector into the cytosol of macrophages.

Comparative Analysis of Different Vaccine Constructs Expressing Defined Antigens from Mycobacterium tuberculosis

BACKGROUND Studies of different vaccine constructs have demonstrated variable efficacy against Mycobacterium tuberculosis in animal models. Despite the fact that these vaccines have used one or another of a very small number of immunodominant antigens, a direct comparison of the relative efficacy of the antigens and delivery systems has been difficult, because the studies have used different parameters for assessment. METHODS We compared the efficacies of the most commonly used vaccine constructs--adjuvanted protein, plasmid DNA, and live bacterial vectors--bearing the immunodominant secreted antigens early secreted antigen target-6 and antigen 85B, either alone or as a fusion protein. Mice were vaccinated with these constructs, and the effects of different delivery systems on protective efficacy (as assessed by survival studies and by monitoring bacterial load) and antigen-specific responses (including the contribution of CD4 and CD8 T cells to these responses) were assayed by various methods. RESULTS The relative efficacy of different vaccines is dependent on the delivery system, the antigen, and the animal model. Likewise, the relative immunodominance of individual antigens in the fusion molecule is altered by the choice of delivery system. CONCLUSION These results clearly demonstrate the importance of assessing vaccine function by use of multiple parameters and indicate which parameters are most reliable for assessing vaccine efficacy.

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.