Replicating viral vector platform exploits alarmin signals for potent CD8+ T cell-mediated tumour immunotherapy

Viral infections lead to alarmin release and elicit potent cytotoxic effector T lymphocyte (CTLeff) responses. Conversely, the induction of protective tumour-specific CTLeff and their recruitment into the tumour remain challenging tasks. Here we show that lymphocytic choriomeningitis virus (LCMV) can be engineered to serve as a replication competent, stably-attenuated immunotherapy vector (artLCMV). artLCMV delivers tumour-associated antigens to dendritic cells for efficient CTL priming. Unlike replication-deficient vectors, artLCMV targets also lymphoid tissue stroma cells expressing the alarmin interleukin-33. By triggering interleukin-33 signals, artLCMV elicits CTLeff responses of higher magnitude and functionality than those induced by replication-deficient vectors. Superior anti-tumour efficacy of artLCMV immunotherapy depends on interleukin-33 signalling, and a massive CTLeff influx triggers an inflammatory conversion of the tumour microenvironment. Our observations suggest that replicating viral delivery systems can release alarmins for improved anti-tumour efficacy. These mechanistic insights may outweigh safety concerns around replicating viral vectors in cancer immunotherapy

TBVAC2020 : advancing tuberculosis vaccines from discovery to clinical development

TBVAC2020 is a research project supported by the Horizon 2020 program of the European Commission (EC). It aims at the discovery and development of novel tuberculosis (TB) vaccines from preclinical research projects to early clinical assessment. The project builds on previous collaborations from 1998 onwards funded through the EC framework programs FP5, FP6, and FP7. It has succeeded in attracting new partners from outstanding laboratories from all over the world, now totaling 40 institutions. Next to the development of novel vaccines, TB biomarker development is also considered an important asset to facilitate rational vaccine selection and development. In addition, TBVAC2020 offers portfolio management that provides selection criteria for entry, gating, and priority settings of novel vaccines at an early developmental stage. The TBVAC2020 consortium coordinated by TBVI facilitates collaboration and early data sharing between partners with the common aim of working toward the development of an effective TB vaccine. Close links with funders and other consortia with shared interests further contribute to this goal

TBVAC2020: Advancing tuberculosis vaccines from discovery to clinical development

TBVAC2020 is a research project supported by the Horizon 2020 program of the European Commission (EC). It aims at the discovery and development of novel tuberculosis (TB) vaccines from preclinical research projects to early clinical assessment. The project builds on previous collaborations from 1998 onwards funded through the EC framework programs FP5, FP6, and FP7. It has succeeded in attracting new partners from outstanding laboratories from all over the world, now totaling 40 institutions. Next to the development of novel vaccines, TB biomarker development is also considered an important asset to facilitate rational vaccine selection and development. In addition, TBVAC2020 offers portfolio management that provides selection criteria for entry, gating, and priority settings of novel vaccines at an early developmental stage. The TBVAC2020 consortium coordinated by TBVI facilitates collaboration and early data sharing between partners with the common aim of working toward the development of an effective TB vaccine. Close links with funders and other consortia with shared interests further contribute to this goal

The Nucleoprotein Is Required for Lymphocytic Choriomeningitis Virus-Based Vaccine Vector Immunogenicity

Recombinant glycoprotein-deficient lymphocytic choriomeningitis virus-based vaccine vectors (rLCMV/ΔGP) are potent CD8(+) T cell inducers. To investigate the underlying molecular requirements, we generated a nucleoprotein-deficient vector counterpart (rLCMV/ΔNP). NP but not GP is a minimal trans-acting factor for viral transcription and genome replication. We found that, unlike rLCMV/ΔGP, rLCMV/ΔNP failed to elicit detectable CD8(+) T cell responses unless NP was trans complemented in a transgenic host. Hence, NP-dependent intracellular gene expression is essential for LCMV vector immunogenicity

Lipoarabinomannan-Responsive Polycytotoxic T Cells Are Associated with Protection in Human Tuberculosis.

RationaleThe development of host-targeted, prophylactic, and therapeutic interventions against tuberculosis requires a better understanding of the immune mechanisms that determine the outcome of infection with Mycobacterium tuberculosis.ObjectivesTo identify T-cell-dependent mechanisms that are protective in tuberculosis.MethodsMulticolor flow cytometry, cell sorting and growth inhibition assays were employed to compare the frequency, phenotype and function of T lymphocytes from bronchoalveolar lavage or the peripheral blood.Measurements and main resultsAt two independent study sites, bronchoalveolar lavage cells from donors with latent tuberculosis infection limited the growth of virulent Mycobacterium tuberculosis more efficiently than those in patients who developed disease. Unconventional, glycolipid-responsive T cells contributed to reduced mycobacterial growth because antibodies to CD1b inhibited this effect by 55%. Lipoarabinomannan was the most potent mycobacterial lipid antigen (activation of 1.3% T lymphocytes) and activated CD1b-restricted T cells that limited bacterial growth. A subset of IFN-γ-producing lipoarabinomannan-responsive T cells coexpressed the cytotoxic molecules perforin, granulysin, and granzyme B, which we termed polycytotoxic T cells. Taking advantage of two well-defined cohorts of subjects latently infected with Mycobacterium tuberculosis or patients who developed active disease after infection, we found a correlation between the frequency of polycytotoxic T cells and the ability to control infection (latent tuberculosis infection, 62%; posttuberculosis patients, 26%).ConclusionsOur data define an unconventional CD8(+) T-cell subset (polycytotoxic T cells) that is based on antigen recognition and function. The results link clinical and mechanistic evidence that glycolipid-responsive, polycytotoxic T cells contribute to protection against tuberculosis

Lipoarabinomannan-Responsive Polycytotoxic T Cells Are Associated with Protection in Human Tuberculosis.

RationaleThe development of host-targeted, prophylactic, and therapeutic interventions against tuberculosis requires a better understanding of the immune mechanisms that determine the outcome of infection with Mycobacterium tuberculosis.ObjectivesTo identify T-cell-dependent mechanisms that are protective in tuberculosis.MethodsMulticolor flow cytometry, cell sorting and growth inhibition assays were employed to compare the frequency, phenotype and function of T lymphocytes from bronchoalveolar lavage or the peripheral blood.Measurements and main resultsAt two independent study sites, bronchoalveolar lavage cells from donors with latent tuberculosis infection limited the growth of virulent Mycobacterium tuberculosis more efficiently than those in patients who developed disease. Unconventional, glycolipid-responsive T cells contributed to reduced mycobacterial growth because antibodies to CD1b inhibited this effect by 55%. Lipoarabinomannan was the most potent mycobacterial lipid antigen (activation of 1.3% T lymphocytes) and activated CD1b-restricted T cells that limited bacterial growth. A subset of IFN-γ-producing lipoarabinomannan-responsive T cells coexpressed the cytotoxic molecules perforin, granulysin, and granzyme B, which we termed polycytotoxic T cells. Taking advantage of two well-defined cohorts of subjects latently infected with Mycobacterium tuberculosis or patients who developed active disease after infection, we found a correlation between the frequency of polycytotoxic T cells and the ability to control infection (latent tuberculosis infection, 62%; posttuberculosis patients, 26%).ConclusionsOur data define an unconventional CD8(+) T-cell subset (polycytotoxic T cells) that is based on antigen recognition and function. The results link clinical and mechanistic evidence that glycolipid-responsive, polycytotoxic T cells contribute to protection against tuberculosis

Lipoarabinomannan-Responsive Polycytotoxic T Cells Are Associated with Protection in Human Tuberculosis

RationaleThe development of host-targeted, prophylactic, and therapeutic interventions against tuberculosis requires a better understanding of the immune mechanisms that determine the outcome of infection with Mycobacterium tuberculosis.ObjectivesTo identify T-cell-dependent mechanisms that are protective in tuberculosis.MethodsMulticolor flow cytometry, cell sorting and growth inhibition assays were employed to compare the frequency, phenotype and function of T lymphocytes from bronchoalveolar lavage or the peripheral blood.Measurements and main resultsAt two independent study sites, bronchoalveolar lavage cells from donors with latent tuberculosis infection limited the growth of virulent Mycobacterium tuberculosis more efficiently than those in patients who developed disease. Unconventional, glycolipid-responsive T cells contributed to reduced mycobacterial growth because antibodies to CD1b inhibited this effect by 55%. Lipoarabinomannan was the most potent mycobacterial lipid antigen (activation of 1.3% T lymphocytes) and activated CD1b-restricted T cells that limited bacterial growth. A subset of IFN-γ-producing lipoarabinomannan-responsive T cells coexpressed the cytotoxic molecules perforin, granulysin, and granzyme B, which we termed polycytotoxic T cells. Taking advantage of two well-defined cohorts of subjects latently infected with Mycobacterium tuberculosis or patients who developed active disease after infection, we found a correlation between the frequency of polycytotoxic T cells and the ability to control infection (latent tuberculosis infection, 62%; posttuberculosis patients, 26%).ConclusionsOur data define an unconventional CD8(+) T-cell subset (polycytotoxic T cells) that is based on antigen recognition and function. The results link clinical and mechanistic evidence that glycolipid-responsive, polycytotoxic T cells contribute to protection against tuberculosis

Gel overlay assay of AZP, PN and cytosol proteins.

<p>Azurophile (AZP), peroxidase-negative (PN) and cytosol proteins were run on AU-PAGE, Coomassie stained (left) and analyzed for antibacterial activity against <i>M. smegmatis</i>. Formation of clearing zone indicates the antimycobacterial activity (right).</p

Survival of <i>M. smegmatis</i> and <i>M. bovis</i> BCG in LPS-stimulated and unstimulated whole neutrophils.

<p>4–9×10⁵/ml <i>M. smegmatis</i> (<b>A</b>) and BCG (<b>B</b>) were incubated with LPS stimulated neutrophils (+) and unstimulated neutrophils (−) for indicated time points. Bacterial survival was determined by CFU assay. Medium containing bacteria alone was used as control. Data shown are from one representative experiment of three individual experiments. Experiments were performed in triplicates; mean ± SD are shown; Significance was referred as ** for P<0.0001.</p

Identification of AZP, PN and cytosol proteins by MALDI-TOF mass spectrometry.

*<p>These proteins were identified from total PN and cytosolic proteins (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050345#pone-0050345-g003" target="_blank">Figure 3</a>).</p