Jes Dietrich

A multistage tuberculosis vaccine that confers efficient protection before and after exposure

All tuberculosis vaccines currently in clinical trials are designed as prophylactic vaccines based on early expressed antigens. We have developed a multistage vaccination strategy in which the early antigens Ag85B and 6-kDa early secretory antigenic target (ESAT-6) are combined with the latency-associated protein Rv2660c (H56 vaccine). In CB6F1 mice we show that Rv2660c is stably expressed in late stages of infection despite an overall reduced transcription. The H56 vaccine promotes a T cell response against all protein components that is characterized by a high proportion of polyfunctional CD4+ T cells. In three different pre‐exposure mouse models, H56 confers protective immunity characterized by a more efficient containment of late-stage infection than the Ag85B-ESAT6 vaccine (H1) and BCG. In two mouse models of latent tuberculosis, we show that H56 vaccination after exposure is able to control reactivation and significantly lower the bacterial load compared to adjuvant control mice.

Exchanging ESAT6 with TB10.4 in an Ag85B Fusion Molecule-Based Tuberculosis Subunit Vaccine: Efficient Protection and ESAT6-Based Sensitive Monitoring of Vaccine Efficacy

Previously we have shown that Ag85B-ESAT-6 is a highly efficient vaccine against tuberculosis. However, because the ESAT-6 Ag is also an extremely valuable diagnostic reagent, finding a vaccine as effective as Ag85B-ESAT-6 that does not contain ESAT-6 is a high priority. Recently, we identified a novel protein expressed by Mycobacterium tuberculosis designated TB10.4. In most infected humans, TB10.4 is strongly recognized, raising interest in TB10.4 as a potential vaccine candidate and substitute for ESAT-6. We have now examined the vaccine potential of this protein and found that vaccination with TB10.4 induced a significant protection against tuberculosis. Fusing Ag85B to TB10.4 produced an even more effective vaccine, which induced protection against tuberculosis comparable to bacillus Calmette-Guérin vaccination and superior to the individual Ag components. Thus, Ag85B-TB10 represents a new promising vaccine candidate against tuberculosis. Furthermore, having now exchanged ESAT-6 for TB10.4, we show that ESAT-6, apart from being an excellent diagnostic reagent, can also be used as a reagent for monitoring vaccine efficacy. This may open a new way for monitoring vaccine efficacy in clinical trials.

CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor.

Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down‐regulated following activation of protein kinase C (PKC). Among other substrates the activated PKC in T cells phosphorylates the CD3 gamma subunit of the TCR. To investigate the role of CD3 gamma phosphorylation in PKC‐mediated TCR down‐regulation, point mutated CD3 gamma cDNA was transfected into the CD3 gamma‐negative T cell line JGN and CD3 gamma transfectants were analysed. Phosphorylation at S126 but not S123 in the cytoplasmic tail of CD3 gamma was required for PKC‐mediated down‐regulation of the TCR. Furthermore, analysis of a series of CD3 gamma truncation mutants indicated that in addition to S126 phosphorylation a motif C‐terminal of S126 was required for TCR down‐regulation. Point mutation analyses confirmed this observation and demonstrated that a membrane‐proximal di‐leucine motif (L131 and L132) in the cytoplasmic tail of CD3 gamma was required for PKC‐mediated TCR down‐regulation in addition to phosphorylation at S126. Incubation of T cells in hypertonic medium known to disrupt normal clathrin lattices severely inhibited PKC‐mediated TCR down‐regulation in non‐mutated T cells, indicating that the TCR was down‐regulated by endocytosis via clathrin coated pits. Based on the present results and previously published observations on intracellular receptor sorting, a general model for intracellular sorting of receptors containing di‐leucine‐ or tyrosine‐based motifs is proposed.

Mucosal Administration of Ag85B-ESAT-6 Protects against Infection with Mycobacterium tuberculosis and Boosts Prior Bacillus Calmette-Guérin Immunity

We have examined the intranasal administration of a vaccine against Mycobacterium tuberculosis (M.tb) consisting of the mucosal adjuvant LTK63 and the Ag Ag85B-ESAT-6. Vaccination with LTK63/Ag85B-ESAT-6 gave a strong and sustained Th1 response mediated by IFN-γ-secreting CD4 cells, which led to long-lasting protection against tuberculosis, equivalent to that observed with bacillus Calmette-Guérin (BCG) or Ag85B-ESAT-6 in dimethyldioctadecylammonium bromide/monophosphoryl lipid A. Because a crucial element of novel vaccine strategies is the ability to boost BCG-derived immunity, we also tested whether LTK63/Ag85B-ESAT-6 could act as a BCG booster vaccine in BCG-vaccinated mice. We found that vaccinating with LTK63/Ag85B-ESAT-6 strongly boosted prior BCG-stimulated immunity. Compared with BCG-vaccinated nonboosted mice, we observed that infection with M.tb led to a significant increase in anti-M.tb-specific CD4 T cells in the lungs of LTK63/Ag85B-ESAT-6-boosted animals. This correlated with a significant increase in the protection against M.tb in LTK63/Ag85B-ESAT-6-boosted mice, compared with BCG-vaccinated animals. Thus, LTK63/Ag85B-ESAT-6 represents an efficient preventive vaccine against tuberculosis with a strong ability to boost prior BCG immunity.

The multistage vaccine H56 boosts the effects of BCG to protect cynomolgus macaques against active tuberculosis and reactivation of latent Mycobacterium tuberculosis infection

It is estimated that one-third of the worlds population is infected with Mycobacterium tuberculosis. Infection typically remains latent, but it can reactivate to cause clinical disease. The only vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is largely ineffective, and ways to enhance its efficacy are being developed. Of note, the candidate booster vaccines currently under clinical development have been designed to improve BCG efficacy but not prevent reactivation of latent infection. Here, we demonstrate that administering a multistage vaccine that we term H56 in the adjuvant IC31 as a boost to vaccination with BCG delays and reduces clinical disease in cynomolgus macaques challenged with M. tuberculosis and prevents reactivation of latent infection. H56 contains Ag85B and ESAT-6, which are two of the M. tuberculosis antigens secreted in the acute phase of infection, and the nutrient stress-induced antigen Rv2660c. Boosting with H56/IC31 resulted in efficient containment of M. tuberculosis infection and reduced rates of clinical disease, as measured by clinical parameters, inflammatory markers, and improved survival of the animals compared with BCG alone. Boosted animals showed reduced pulmonary pathology and extrapulmonary dissemination, and protection correlated with a strong recall response against ESAT-6 and Rv2660c. Importantly, BCG/H56-vaccinated monkeys did not reactivate latent infection after treatment with anti-TNF antibody. Our results indicate that H56/IC31 boosting is able to control late-stage infection with M. tuberculosis and contain latent tuberculosis, providing a rationale for the clinical development of H56.

The adjuvant mechanism of cationic dimethyldioctadecylammonium liposomes

Cationic liposomes are being used increasingly as efficient adjuvants for subunit vaccines but their precise mechanism of action is still unknown. Here, we investigated the adjuvant mechanism of cationic liposomes based on the synthetic amphiphile dimethyldioctadecylammonium (DDA). The liposomes did not have an effect on the maturation of murine bone‐marrow‐derived dendritic cells (BM‐DCs) related to the surface expression of major histocompatibility complex (MHC) class II, CD40, CD80 and CD86. We found that ovalbumin (OVA) readily associated with the liposomes (> 90%) when mixed in equal concentrations. This efficient adsorption onto the liposomes led to an enhanced uptake of OVA by BM‐DCs as assessed by flow cytometry and confocal fluorescence laser‐scanning microscopy. This was an active process, which was arrested at 4° and by an inhibitor of actin‐dependent endocytosis, cytochalasin D. In vivo studies confirmed the observed effect because adsorption of OVA onto DDA liposomes enhanced the uptake of the antigen by peritoneal exudate cells after intraperitoneal injection. The liposomes targeted antigen preferentially to antigen‐presenting cells because we only observed a minimal uptake by T cells in mixed splenocyte cultures. The adsorption of antigen onto the liposomes increased the efficiency of antigen presentation more than 100 times in a responder assay with MHC class II‐restricted OVA‐specific T‐cell receptor transgenic DO11.10 T cells. Our data therefore suggest that the primary adjuvant mechanism of cationic DDA liposomes is to target the cell membrane of antigen‐presenting cells, which subsequently leads to enhanced uptake and presentation of antigen.

Ig-Like Transcript 2 (ILT2)/Leukocyte Ig-Like Receptor 1 (LIR1) Inhibits TCR Signaling and Actin Cytoskeleton Reorganization

Ig-like transcript 2 (ILT2)/leukocyte Ig-like receptor 1 (LIR1) is a receptor, specific for MHC class I molecules, that inhibits lymphoid and myeloid cells. Here, we analyzed the molecular and cellular mechanisms by which ILT2 modulates T cell activation in primary CTLs and transfected T cell lines. We found that cross-linking with the TCR and the activity of Src tyrosine kinase p56lck were required for phosphorylation of ILT2 and subsequent recruitment of Src homology protein 1. In contrast, ILT2 triggering resulted in reduced phosphorylation of TCRζ and linker for activation of T cells, which led to reduced TCRζ-ZAP70 complex formation, as well as extracellular signal-related kinase 1 and 2 activation. Furthermore, ILT2 inhibited both superantigen and anti-TCR Ab-induced rearrangement of the actin cytoskeleton. The inhibitory effect mediated by ILT2 is probably concentrated at the APC-T cell interface because both TCR and ILT2 were strongly polarized toward the APC upon engagement by their specific ligands. Thus, ILT2 inhibits both signaling and cellular events involved in the activation of T cells.

Protection and Polyfunctional T Cells Induced by Ag85B-TB10.4/IC31® against Mycobacterium tuberculosis Is Highly Dependent on the Antigen Dose

Background Previously we have shown that Ag85B-TB10.4 is a highly efficient vaccine against tuberculosis when delivered in a Th1 inducing adjuvant based on cationic liposomes. Another Th1 inducing adjuvant, which has shown a very promising profile in both preclinical and clinical trials, is IC31®. In this study, we examined the potential of Ag85B-TB10.4 delivered in the adjuvant IC31® for the ability to induce protection against infection with Mycobacterium tuberculosis. In addition, we examined if the antigen dose could influence the phenotype of the induced T cells. Methods and Findings We found that vaccination with the combination of Ag85B-TB10.4 and IC31® resulted in high numbers of polyfunctional CD4 T cells co-expressing IL-2, IFN-γ and TNF-α. This correlated with protection against subsequent challenge with M.tb in the mouse TB model. Importantly, our results also showed that both the vaccine induced T cell response, and the protective efficacy, was highly dependent on the antigen dose. Thus, whereas antigen doses of 5 and 15 µg did not induce significant protection against M.tb, reducing the dose to 0.5 µg selectively increased the number of polyfunctional T cells and induced a strong protection against infection with M.tb. The influence of antigen dose was also observed in the guinea pig model of aerosol infection with M.tb. In this model a 2.5 fold increase in the antigen dose reduced the protection against infection with M.tb to the level observed in non-vaccinated animals. Conclusions/Significance Small changes in the antigen dose can greatly influence the induction of specific T cell subpopulations and the dose is therefore a crucial factor when testing new vaccines. However, the adjuvant IC31® can, with the optimal dose of Ag85B-TB10.4, induce strong protection against Mycobacterium tuberculosis. This vaccine has now entered clinical trials.

TB vaccines: current status and future perspectives

Vaccines against intracellular pathogens such as Mycobacterium tuberculosis need to induce strong cellular immune responses. Antigen discovery programs have exploited this and used proteome studies and T‐cell recognition in PPD‐positive individuals to select proteins and after testing for protective efficacy in animals the most promising proteins have been put together in fusion molecules. Three such fusion proteins are currently in clinical trials, the two most advanced have already passed phase I trials and are entering phase II.

Induction of CD8 T Cells against a Novel Epitope in TB10.4: Correlation with Mycobacterial Virulence and the Presence of a Functional Region of Difference-1

Although infection with Mycobacterium tuberculosis (M.tb) induces a robust CD8 T cell response, the role of CD8 T cells in the defense against M.tb, and the mechanisms behind the induction of CD8 T cells, is still not clear. TB10.4 is a recently described Ag that is expressed by both bacillus Calmette-Guérin (BCG) and M.tb. In the present study, we describe a novel CD8 T cell epitope in TB10.4, TB10.43-11. We show that TB10.43-11-specific CD8 T cells are induced at the onset of infection and are present throughout the infection in high numbers. TB10.43-11 CD8 T cells were recruited to the site of infection and expressed CD44, TNF-α, and IFN-γ. In addition, TB10.43-11 CD8 T cells showed an up-regulation of FasL and LAMP-1/2 (CD107A/B), which correlated with a strong in vivo cytolytic activity. The induction of TB10.43-11-specific CD8 T cells was less pronounced following infection with BCG compared to infection with M.tb. By using a rBCG expressing the genetic region of difference-1 (RD1), we show that the presence of a functional RD1 region increases the induction of TB10.43-11-specific CD8 T cells as well as the bacterial virulence. Finally, as an M.tb variant lacking the genetic region RD1 also induced a significant amount of TB10.43-11-specific CD8 T cells, and exhibited increased virulence compared with BCG, our data suggest that virulence in itself is also involved in generating a robust CD8 T cell response against mycobacterial epitopes, such as TB10.43-11.