Intracellular bacteria as DNA carriers in vitro and in vivo

In the first part of this work, the mechanisms involved in plasmid transfer from the intracellular bacterium L. monocytogenes to eukaryotic host cells were investigated. It could be shown that the initial invasion of the host cell is not limiting for efficient gene transfer. Also, bacteria are efficiently lysed upon antibiotic treatment and release their content to the cytosol, but only a fraction of the plasmids is eventually transferred to the nucleus. Released plasmids are probably associated with high molecular weight components which might limit nuclear transfer. Also, a small amount of bacterial chromosomal DNA was transferred to the nucleus and integrated in the host cell genome. In the second part of this work, L. monocytogenes and S. flexneri were tested as gene transfer vehicles for cancer therapy. The dissemination of both bacteria was analyzed after infection via the intratumoural route. The bacteria were not contained within the tumour tissue but were also found in liver and spleen of mice. When mice were systemically infected with attenuated S. flexneri, the bacteria were rapidly cleared from the blood stream and did not accumulate in tumours or organs, as described before for other bacterial species. Gene transfer from bacteria to tumour cells in vivo could not clearly be demonstrated for Listeria under the conditions used. In contrast, some implication of gene transfer by Shigella was found. Here, refinement of the technical settings should allow the definite detection of even low levels of transgene expression. In summary, extending the present work will result in better gene transfer abilities and will reveal the great potential of such bacteria as DNA carriers.Im ersten Teil dieser Arbeit wurden die Mechanismen untersucht, die dem Plasmidtransfer vom intrazellulären Bakterium Listeria monocytogenes in eukaryontische Wirtszellen zugrunde liegen. Es konnte gezeigt werden, dass die Invasion der Wirtszelle nicht limitierend für effizienten Gentransfer ist. Nach Antibiotika-Behandlung lysieren die intrazellulären Bakterien vollständig und setzen ihren Inhalt im Wirtszellzytosol frei, jedoch erreicht nur ein Teil der Plasmide letztlich den Zellkern. Freigesetzte Plasmide sind wahrscheinlich mit Partikeln von hohem Molekulargewicht assoziiert, die den Kerntransfer behindern könnten. Auch ein kleiner Teil bakterieller chromosomaler DNA wurde in den Kern transferiert und in das Wirtszellgenom integriert. Im zweiten Teil dieser Arbeit wurden L. monocytogenes und S. flexneri als Gentransfervektoren für die Krebstherapie untersucht. Die Verteilung beider Stämme wurde nach intratumoraler Verabreichung analysiert. Die Bakterien waren nicht im Tumorgewebe eingeschlossen, sondern wurden auch in Milz und Leber der Mäuse gefunden. Nach systemischer Infektion von Mäusen mit S. flexneri wurden die Bakterien schnell aus dem Blut entfernt und akkumulierten nicht im Tumorgewebe, wie für einige andere bakterielle Spezies beschrieben. Unter den hier angewandten Bedingungen konnte in vivo Gentransfer in Tumorzellen durch Listerien nicht zweifelsfrei gezeigt werden. Einige Anzeichen für Gentransfer durch Shigellen wurden jedoch gefunden. Eine Verfeinerung der angewandten Techniken sollte hier die Detektion auch von niedriger Transgenexpression erlauben. Zusammenfassend wird die Fortführung der hier präsentierten Arbeit zu einer Verbesserung des Gentransfers führen und das große Potential solcher Bakterien als DNA Überträger aufdecken

Demonstrating the utility of the ex vivo murine mycobacterial growth inhibition assay (MGIA) for high-throughput screening of tuberculosis vaccine candidates against multiple Mycobacterium tuberculosis complex strains

Human tuberculosis (TB) is caused by various members of the Mycobacterium tuberculosis (Mtb) complex. Differences in host response to infection have been reported, illustrative of a need to evaluate efficacy of novel vaccine candidates against multiple strains in preclinical studies. We previously showed that the murine lung and spleen direct mycobacterial growth inhibition assay (MGIA) can be used to assess control of ex vivo mycobacterial growth by host cells. The number of mice required for the assay is significantly lower than in vivo studies, facilitating testing of multiple strains and/or the incorporation of other cellular analyses. Here, we provide proof-of-concept that the murine MGIA can be applied to evaluate vaccine-induced protection against multiple Mtb clinical isolates. Using an ancient and modern strain of the Mtb complex, we demonstrate that ex vivo bacillus Calmette–Guérin (BCG)-mediated mycobacterial growth inhibition recapitulates protection observed in the lung and spleen following in vivo infection of mice. Further, we provide the first report of cellular and transcriptional correlates of BCG-induced growth inhibition in the lung MGIA. The ex vivo MGIA represents a promising platform to gain early insight into vaccine performance against a collection of Mtb strains and improve preclinical evaluation of TB vaccine candidates

Assessment of the reproducibility and inter-site transferability of the murine direct splenocyte mycobacterial growth inhibition assay (MGIA)

Tuberculosis (TB) vaccine candidates must be tested for safety and efficacy using preclinical challenge models prior to advancement to human trials, because of the lack of a validated immune correlate or biomarker of protection. New, unbiased tools are urgently needed to expedite the selection of vaccine candidates at an early stage of development and reduce the number of animals experimentally infected with virulent Mycobacterium tuberculosis (M.tb). In recent years, there has been a concerted effort to develop standardised functional ex vivo mycobacterial growth inhibition assays (MGIAs) as a potential surrogate read-out of vaccine efficacy. We have previously described a direct MGIA for use with mouse splenocytes. In the current study, we set out to systematically compare co-culture conditions for the murine direct splenocyte MGIA with respect to both intra-assay repeatability and inter-site reproducibility. Common sample sets were shared between laboratory sites and reproducibility and sensitivity to detect a BCG-vaccine induced response were assessed. Co-culturing 5×106 splenocytes in 48-well plates resulted in improved reproducibility and superior sensitivity to detect a vaccine response compared with standing or rotating sealed 2ml screw-cap tubes. As the difference between naïve and BCG vaccinated mice was not consistently detected across both sample sets at both sites, we sought to further improve assay sensitivity by altering the multiplicity of infection (MOI). Cell viability at the end of the co-culture period was improved when splenocyte input number was reduced, with the highest viability for the condition of 3×106 splenocytes in 48-well plates. This cell input was also associated with the greatest sensitivity to detect a BCG vaccine-mediated MGIA response using an M.tb inoculum. Based on our findings, we recommend optimal co-culture conditions in a move towards aligning direct MGIA protocols and generating a cross-species consensus for early evaluation of TB vaccine candidates and biomarker studies

High monocyte to lymphocyte ratio is associated with impaired protection after subcutaneous administration of BCG in a mouse model of tuberculosis.

Background: The only available tuberculosis (TB) vaccine, Bacillus Calmette-Guérin (BCG), has variable efficacy. New vaccines are therefore urgently needed. Why BCG fails is incompletely understood, and the tools used for early assessment of new vaccine candidates do not account for BCG variability. Taking correlates of risk of TB disease observed in human studies and back-translating them into mice to create models of BCG variability should allow novel vaccine candidates to be tested early in animal models that are more representative of the human populations most at risk. Furthermore, this could help to elucidate the immunological mechanisms leading to BCG failure. We have chosen the monocyte to lymphocyte (ML) ratio as a correlate of risk of TB disease and have back-translated this into a mouse model. Methods: Four commercially available, inbred mouse strains were chosen. We investigated their baseline ML ratio by flow cytometry; extent of BCG-mediated protection from M ycobacterium tuberculosis infection by experimental challenge; vaccine-induced interferon gamma (IFNγ) response by ELISPOT assay; and tissue distribution of BCG by plating tissue homogenates. Results: The ML ratio varied significantly between A/J, DBA/2, C57Bl/6 and 129S2 mice. A/J mice showed the highest BCG-mediated protection and lowest ML ratio, while 129S2 mice showed the lowest protection and higher ML ratio. We also found that A/J mice had a lower antigen specific IFNγ response than 129S2 mice. BCG tissue distribution appeared higher in A/J mice, although this was not statistically significant. Conclusions: These results suggest that the ML ratio has an impact on BCG-mediated protection in mice, in alignment with observations from clinical studies. A/J and 129S2 mice may therefore be useful models of BCG vaccine variability for early TB vaccine testing. We speculate that failure of BCG to protect from TB disease is linked to poor tissue distribution in a ML high immune environment

The Cross-Species Mycobacterial Growth Inhibition Assay (MGIA) Project, 2010-2014.

The development of a functional biomarker assay in the tuberculosis (TB) field would be widely recognized as a major advance in efforts to develop and to test novel TB vaccine candidates efficiently. We present preliminary studies using mycobacterial growth inhibition assays (MGIAs) to detect Mycobacterium bovis BCG vaccine responses across species, and we extend this work to determine whether a standardized MGIA can be applied in characterizing new TB vaccines. The comparative MGIA studies reviewed here aimed to evaluate robustness, reproducibility, and ability to reflect in vivo responses. In doing so, they have laid the foundation for the development of a MGIA that can be standardized and potentially qualified. A major challenge ahead lies in better understanding the relationships between in vivo protection, in vitro growth inhibition, and the immune mechanisms involved. The final outcome would be a MGIA that could be used with confidence in TB vaccine trials. We summarize data arising from this project, present a strategy to meet the goals of developing a functional assay for TB vaccine testing, and describe some of the challenges encountered in performing and transferring such assays

Ex vivo mycobacterial growth inhibition assay (MGIA) for tuberculosis vaccine testing - a protocol for mouse splenocytes

The testing of vaccines for tuberculosis is costly and time-consuming, and dependent on preclinical animal challenge models and clinical trials. We have recently developed a mycobacterial growth inhibition assay (MGIA) to test vaccine efficacy ex vivo. This assay measures the summative effect of the host immune response and may serve as a novel tool to facilitate vaccine testing. It has generated much interest recently, and to facilitate technology transfer and reproducibility between laboratories, we here describe a detailed protocol for an ex vivo MGIA in mouse splenocytes.</jats:p

In vitro Mycobacterial Growth Inhibition in South Korean Adults With Latent TB Infection

It is important to understand the ability to inhibit mycobacterial growth in healthy adults who would have been Bacillus Calmette-Guérin (BCG) vaccinated in childhood as this group will be the potential target population for novel booster TB vaccine trials. In this study we investigated not only the long-term immunity induced by childhood BCG vaccination but also protective immunity in terms of the ability to inhibit mycobacterial growth in those who were BCG vaccinated in childhood, with evidence of recent or remote TB infection.This study was supported by a grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF) founded by the Ministry of Science, ICT and Future Planning (NRF-2015K1A3A7A03073714) and from the Korean Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) founded by the Ministry for Health, Welfare, and Family Affairs, Republic of Korea (HI14C1324). LSHTM and ITRC are partners in a grant under the MRC-KHIDI UK-Korea Partnering Award scheme awarded to HL and SS with Grant No. HI17C0324 and MC_PC_17109 in TBVAC2020 supported by the European Commission under the H2020 program, with Grant No. 643381; the ITRC group was funded by a Grant (NRF-2015K1A3A7A03073714)

Polyfunctional CD4 T-cells correlate with in vitro mycobacterial growth inhibition following Mycobacterium bovis BCG-vaccination of infants

Background: Vaccination with Bacillus Calmette Guerin (BCG) protects infants against childhood tuberculosis however the immune mechanisms involved are not well understood. Further elucidation of the infant immune response to BCG will aid with the identification of immune correlates of protection against tuberculosis and with the design of new improved vaccines. The purpose of this study was to investigate BCG-induced CD4+ T-cell responses in blood samples from infants for cytokine secretion profiles thought to be important for protection against tuberculosis and compare these to PBMC-mediated in vitro mycobacterial growth inhibition. Methods: Blood from BCG-vaccinated or unvaccinated infants was stimulated overnight with Mycobacterium tuberculosis (M. tb) purified protein derivative (PPD) or controls and intracellular cytokine staining and flow cytometry used to measure CD4+ T-cell responses. PBMC cryopreserved at the time of sample collection were thawed and incubated with live BCG for four days following which inhibition of BCG growth was determined. Results: PPD-specific IFNc+TNFa+IL-2+CD4+ T-cells represented the dominant T-cell response at 4 months and 1 year after infant BCG. These responses were undetectable in age-matched unvaccinated infants. IL-17+ CD4+ T-cells were significantly more frequent in vaccinated infants at 4 months but not at 1-year post-BCG. PBMC-mediated inhibition of mycobacterial growth was significantly enhanced at 4 months post-BCG as compared to unvaccinated controls. In an analysis of all samples with both datasets available, mycobacterial growth inhibition correlated significantly with the frequency of polyfunctional (IFNc+TNFa+IL-2+) CD4+ T-cells. Conclusions: These data suggest that BCG vaccination of infants induces specific polyfunctional T-helper-1 and T-helper-17 responses and the ability, in the PBMC compartment, to inhibit the growth of mycobacteria in vitro. We also demonstrate that polyfunctional T-helper-1 cells may play a role in growth inhibition as evidenced by a significant correlation between the two.This work was supported by the European Commission within the 7th framework program (FP7) NEWTBVAC (Grant No. HEALTH-F3-2009-241745), by the European Tuberculosis Vaccine Initiative (Grant No. TBVI-12 NORAD) and by the European Commission within Horizon2020 TBVAC2020 (Grant No. H2020 PHC- 643381)

Noninvasive biophotonic imaging for studies of infectious disease

According to World Health Organization estimates, infectious organisms are responsible for approximately one in four deaths worldwide. Animal models play an essential role in the development of vaccines and therapeutic agents but large numbers of animals are required to obtain quantitative microbiological data by tissue sampling. Biophotonic imaging (BPI) is a highly sensitive, nontoxic technique based on the detection of visible light, produced by luciferase-catalysed reactions (bioluminescence) or by excitation of fluorescent molecules, using sensitive photon detectors. The development of bioluminescent/fluorescent microorganisms therefore allows the real-time noninvasive detection of microorganisms within intact living animals. Multiple imaging of the same animal throughout an experiment allows disease progression to be followed with extreme accuracy, reducing the number of animals required to yield statistically meaningful data. In the study of infectious disease, the use of BPI is becoming widespread due to the novel insights it can provide into established models, as well as the impact of the technique on two of the guiding principles of using animals in research, namely reduction and refinement. Here, we review the technology of BPI, from the instrumentation through to the generation of a photonic signal, and illustrate how the technique is shedding light on infection dynamics in vivo