Susan J. F. van den Eeden

Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1.

With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome–lysosome fusion through the AS160 (also known as TBC1D4)–RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.

CD8+ CTL Priming by Exact Peptide Epitopes in Incomplete Freund’s Adjuvant Induces a Vanishing CTL Response, whereas Long Peptides Induce Sustained CTL Reactivity

Therapeutic vaccination trials, in which patients with cancer were vaccinated with minimal CTL peptide in oil-in-water formulations, have met with limited success. Many of these studies were based on the promising data of mice studies, showing that vaccination with a short synthetic peptide in IFA results in protective CD8+ T cell immunity. By use of the highly immunogenic OVA CTL peptide in IFA as a model peptide-based vaccine, we investigated why minimal CTL peptide vaccines in IFA performed so inadequately to allow full optimization of peptide vaccination. Injection of the minimal MHC class I-binding OVA257–264 peptide in IFA transiently activated CD8+ effector T cells, which eventually failed to undergo secondary expansion or to kill target cells, as a result of a sustained and systemic presentation of the CTL peptides gradually leaking out of the IFA depot without systemic danger signals. Complementation of this vaccine with the MHC class II-binding Th peptide (OVA323–339) restored both secondary expansion and in vivo effector functions of CD8+ T cells. Simply extending the CTL peptide to a length of 30 aa also preserved these CD8+ T cell functions, independent of T cell help, because the longer CTL peptide was predominantly presented in the locally inflamed draining lymph node. Importantly, these functional differences were reproduced in two additional model Ag systems. Our data clearly show why priming of CTL with minimal peptide epitopes in IFA is suboptimal, and demonstrate that the use of longer versions of these CTL peptide epitopes ensures the induction of sustained effector CD8+ T cell reactivity in vivo.

Superior induction of anti-tumor CTL immunity by extended peptide vaccines involves prolonged, DC-focused antigen presentation.

Anti‐tumor vaccines consisting of extended CTL peptides in combination with CpG‐ODN were shown to be superior to those comprising minimal CTL epitopes and CpG‐ODN, in that they elicit stronger effector CTL responses with greater tumoricidal potential. We now demonstrate that this improved performance is primarily due to the focusing of CTL epitope presentation onto activated DC in the inflamed lymph nodes draining the vaccination site. In the case of vaccination with minimal peptides, additional APC including T and B cells are also loaded with CTL epitopes. Our data suggest that circulation of these peptide‐loaded lymphocytes leads to epitope presentation in non‐inflamed lymphoid organs distal from the vaccination site, in the absence of potent costimulatory signals required for efficient CTL priming. The resulting blend of pro‐immunogenic and tolerogenic signals, which results in suboptimal activation of the CTL response, is avoided by vaccinating with extended CTL peptides. An additional advantage of extended CTL peptide vaccines is an increased duration of in vivo epitope presentation.

Detection of human papillomavirus type 18 E6 and E7-specific CD4+ T-helper 1 immunity in relation to health versus disease.

The most common high‐risk human papillomavirus types, HPV16 and 18, differ markedly with respect to their interaction with the host. Clearance of HPV18 infections generally takes longer and HPV18‐positive cancers have a poorer prognosis. We therefore evaluated Th1‐type immunity against the E6 and E7 oncoproteins of HPV18 in healthy subjects and in patients with HPV18‐positive genital cancer, and compared the results to our previously obtained data for HPV16. Approximately 20% of the healthy individuals displayed immunity against HPV18 E6. In contrast, none of the patients showed such responses, despite the presence of HPV18‐positive lesions. Several of the patients did respond to HPV18 E7, whereas this immunity is rarely found in healthy subjects. This pattern of immune reactivity is essentially similar to that previously found for HPV16. It is unlikely that this similarity is the result of immunological cross‐reactivity between the E6 and E7 antigens of HPV types 16 and 18. Our data confirm the relation between failure of E6‐specific Th1 immunity and high‐risk HPV‐induced cervical neoplasia and argue that parameters other than these determine the differences in pathological impact between HPV types 16 and 18.

An Unbiased Genome-Wide Mycobacterium tuberculosis Gene Expression Approach To Discover Antigens Targeted by Human T Cells Expressed during Pulmonary Infection

Mycobacterium tuberculosis is responsible for almost 2 million deaths annually. Mycobacterium bovis bacillus Calmette-Guérin, the only vaccine available against tuberculosis (TB), induces highly variable protection against TB, and better TB vaccines are urgently needed. A prerequisite for candidate vaccine Ags is that they are immunogenic and expressed by M. tuberculosis during infection of the primary target organ, that is, the lungs of susceptible individuals. In search of new TB vaccine candidate Ags, we have used a genome-wide, unbiased Ag discovery approach to investigate the in vivo expression of 2170 M. tuberculosis genes during M. tuberculosis infection in the lungs of mice. Four genetically related but distinct mouse strains were studied, representing a spectrum of TB susceptibility controlled by the supersusceptibility to TB 1 locus. We used stringent selection approaches to select in vivo–expressed M. tuberculosis (IVE-TB) genes and analyzed their expression patterns in distinct disease phenotypes such as necrosis and granuloma formation. To study the vaccine potential of these proteins, we analyzed their immunogenicity. Several M. tuberculosis proteins were recognized by immune cells from tuberculin skin test-positive, ESAT6/CFP10-responsive individuals, indicating that these Ags are presented during natural M. tuberculosis infection. Furthermore, TB patients also showed responses toward IVE-TB Ags, albeit lower than tuberculin skin test-positive, ESAT6/CFP10-responsive individuals. Finally, IVE-TB Ags induced strong IFN-γ+/TNF-α+ CD8+ and TNF-α+/IL-2+ CD154+/CD4+ T cell responses in PBMC from long-term latently M. tuberculosis–infected individuals. In conclusion, these IVE-TB Ags are expressed during pulmonary infection in vivo, are immunogenic, induce strong T cell responses in long-term latently M. tuberculosis–infected individuals, and may therefore represent attractive Ags for new TB vaccines.

Field-Evaluation of a New Lateral Flow Assay for Detection of Cellular and Humoral Immunity against Mycobacterium leprae

Background Field-applicable tests detecting asymptomatic Mycobacterium leprae (M. leprae) infection or predicting progression to leprosy, are urgently required. Since the outcome of M. leprae infection is determined by cellular- and humoral immunity, we aim to develop diagnostic tests detecting pro-/anti-inflammatory and regulatory cytokines as well as antibodies against M. leprae. Previously, we developed lateral flow assays (LFA) for detection of cytokines and anti-PGL-I antibodies. Here we evaluate progress of newly developed LFAs for applications in resource-poor settings. Methods The combined diagnostic value of IP-10, IL-10 and anti-PGL-I antibodies was tested using M. leprae-stimulated blood of leprosy patients and endemic controls (EC). For reduction of the overall test-to-result time the minimal whole blood assay time required to detect distinctive responses was investigated. To accommodate LFAs for field settings, dry-format LFAs for IP-10 and anti-PGL-I antibodies were developed allowing storage and shipment at ambient temperatures. Additionally, a multiplex LFA-format was applied for simultaneous detection of anti-PGL-I antibodies and IP-10. For improved sensitivity and quantitation upconverting phosphor (UCP) reporter technology was applied in all LFAs. Results Single and multiplex UCP-LFAs correlated well with ELISAs. The performance of dry reagent assays and portable, lightweight UCP-LF strip readers indicated excellent field-robustness. Notably, detection of IP-10 levels in stimulated samples allowed a reduction of the whole blood assay time from 24 h to 6 h. Moreover, IP-10/IL-10 ratios in unstimulated plasma differed significantly between patients and EC, indicating the feasibility to identify M. leprae infection in endemic areas. Conclusions Dry-format UCP-LFAs are low-tech, robust assays allowing detection of relevant cytokines and antibodies in response to M. leprae in the field. The high levels of IP-10 and the required shorter whole blood assay time, render this cytokine useful to discriminate between leprosy patients and EC.

Longitudinal immune profiles in type 1 leprosy reactions in Bangladesh, Brazil, Ethiopia and Nepal.

BackgroundAcute inflammatory reactions are a frequently occurring, tissue destructing phenomenon in infectious- as well as autoimmune diseases, providing clinical challenges for early diagnosis. In leprosy, an infectious disease initiated by Mycobacterium leprae (M. leprae), these reactions represent the major cause of permanent neuropathy. However, laboratory tests for early diagnosis of reactional episodes which would significantly contribute to prevention of tissue damage are not yet available.Although classical diagnostics involve a variety of tests, current research utilizes limited approaches for biomarker identification. In this study, we therefore studied leprosy as a model to identify biomarkers specific for inflammatory reactional episodes.MethodsTo identify host biomarker profiles associated with early onset of type 1 leprosy reactions, prospective cohorts including leprosy patients with and without reactions were recruited in Bangladesh, Brazil, Ethiopia and Nepal. The presence of multiple cyto-/chemokines induced by M. leprae antigen stimulation of peripheral blood mononuclear cells as well as the levels of antibodies directed against M. leprae-specific antigens in sera, were measured longitudinally in patients.ResultsAt all sites, longitudinal analyses showed that IFN-γ-, IP-10-, IL-17- and VEGF-production by M. leprae (antigen)-stimulated PBMC peaked at diagnosis of type 1 reactions, compared to when reactions were absent. In contrast, IL-10 production decreased during type 1 reaction while increasing after treatment. Thus, ratios of these pro-inflammatory cytokines versus IL-10 provide useful tools for early diagnosing type 1 reactions and evaluating treatment. Of further importance for rapid diagnosis, circulating IP-10 in sera were significantly increased during type 1 reactions. On the other hand, humoral immunity, characterized by M. leprae-specific antibody detection, did not identify onset of type 1 reactions, but allowed treatment monitoring instead.ConclusionsThis study identifies immune-profiles as promising host biomarkers for detecting intra-individual changes during acute inflammation in leprosy, also providing an approach for other chronic (infectious) diseases to help early diagnose these episodes and contribute to timely treatment and prevention of tissue damage.

New Genome-Wide Algorithm Identifies Novel In-Vivo Expressed Mycobacterium Tuberculosis Antigens Inducing Human T-Cell Responses with Classical and Unconventional Cytokine Profiles

New strategies are needed to develop better tools to control TB, including identification of novel antigens for vaccination. Such Mtb antigens must be expressed during Mtb infection in the major target organ, the lung, and must be capable of eliciting human immune responses. Using genome-wide transcriptomics of Mtb infected lungs we developed data sets and methods to identify IVE-TB (in-vivo expressed Mtb) antigens expressed in the lung. Quantitative expression analysis of 2,068 Mtb genes from the predicted first operons identified the most upregulated IVE-TB genes during in-vivo pulmonary infection. By further analysing high-level conservation among whole-genome sequenced Mtb-complex strains (n = 219) and algorithms predicting HLA-class-Ia and II presented epitopes, we selected the most promising IVE-TB candidate antigens. Several of these were recognized by T-cells from in-vitro Mtb-PPD and ESAT6/CFP10-positive donors by proliferation and multi-cytokine production. This was validated in an independent cohort of latently Mtb-infected individuals. Significant T-cell responses were observed in the absence of IFN-γ-production. Collectively, the results underscore the power of our novel antigen discovery approach in identifying Mtb antigens, including those that induce unconventional T-cell responses, which may provide important novel tools for TB vaccination and biomarker profiling. Our generic approach is applicable to other infectious diseases.

Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis

ABSTRACT Responsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions. Mycobacterium bovis BCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity against Mycobacterium tuberculosis latency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a major M. tuberculosis latency antigen which is highly expressed by “dormant” M. tuberculosis and well recognized by T cells from latently M. tuberculosis-infected individuals. In order to assess its in vivo immunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ+/TNF+) and IFN-γ+ CD4+ T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs of M. tuberculosis-challenged mice. This was observed both in a pre- and in a post-M. tuberculosis challenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential of M. tuberculosis latency antigens to improve BCG efficacy. These data suggest a promising role for M. tuberculosis latency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting.

The in vivo expressed Mycobacterium tuberculosis (IVE-TB) antigen Rv2034 induces CD4⁺ T-cells that protect against pulmonary infection in HLA-DR transgenic mice and guinea pigs.

Tuberculosis (TB) remains a life-threatening infectious disease of global proportions with serious negative health and economic consequences. The lack of sufficient protection induced by Mycobacterium bovis BCG, the current vaccine for TB, as well as the impact of HIV co-infection and the emergence of drug resistant Mycobacterium tuberculosis (Mtb) strains all urge for improved vaccines against TB. A minimal requirement for Mtb vaccine antigens is their in vivo expression during Mtb infection and ability to trigger significant immune responses. Recently we identified a new class of Mtb antigens, designated IVE-TB (in vivo expressed) antigens. These included Rv2034, a protein that was expressed during pulmonary infection and strongly recognized by human T-cells. Here, the in vivo immunogenicity and protective efficacy of Rv2034 was further analyzed using HLA-DR transgenic mice that lack endogenous murine MHC class II molecules. The Rv2034 protein indeed was highly immunogenic in HLA-DR3 transgenic mice and induced HLA-DR3 restricted IFN-γ(+)/TNF(+) and IFN-γ(+) CD4(+) T-cells, specific for an epitope encoded in peptide 31-50. CD4(+) T-cell responses were optimally induced when using TLR9- and TLR3-ligand-adjuvants or CAF09. Rv2034-specific antibodies were observed following immunization with either TLR2-, TLR3-, TLR4-, TLR5-, TLR7- or TLR9-ligands or CAF09. Importantly, immunization with Rv2034 or the hybrid-protein Ag85B-ESAT6-Rv2034 adjuvanted with CpG or CAF09, induced over one log reduction, relative to unvaccinated controls, in the number of bacilli in the lungs of Mtb challenged HLA-DR3 transgenic mice and guinea pigs. These data demonstrate the potential of Rv2034 as a novel, IVE-TB antigen for future TB vaccination.