Claus Aagaard

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.

Tuberculosis Subunit Vaccination Provides Long-Term Protective Immunity Characterized by Multifunctional CD4 Memory T Cells

Improved vaccines capable of promoting long-term cellular immunity are urgently required for a number of diseases that remain global health problems. In the present study, we demonstrate that a tuberculosis subunit vaccine, Ag85B-ESAT-6/CAF01 (where ESAT-6 is early secreted antigenic target of 6 kDa and CAF01 is cationic adjuvant formulation 01), induces very robust memory CD4 T cell responses that are maintained at high levels for >1 year postvaccination. This long-term, vaccine-induced memory response protects against a challenge with Mycobacterium tuberculosis at levels that are comparable to or better than those of bacillus Calmette-Guérin. Characterization of the CD4 memory T cells by multicolor flow cytometry demonstrated that the long-lived memory population consisted almost exclusively of TNF-α+IL-2+ and IFN-γ+TNF-α+IL-2+ multifunctional T cells. In addition, memory cells isolated >1 year postvaccination maintained a strong, vaccine-specific proliferative potential. Long-term memory induced by the BCG vaccine contained fewer multifunctional T cells and was biased toward effector cells mainly of the TNF-α+IFN-γ+-coexpressing subset. Ag85B-ESAT-6/CAF01 vaccination very efficiently sustained multifunctional CD4 T cells that accumulated at the site of infection after M. tuberculosis challenge, whereas the response in unvaccinated animals was characterized by CD4 effector T cells. Our data demonstrate that adjuvanted subunit vaccines can promote long-term protective immune responses characterized by high levels of persisting multifunctional T cells and that the quality and profile of this response is sustained postinfection.

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.

Cationic Liposomes Formulated with Synthetic Mycobacterial Cordfactor (CAF01): A Versatile Adjuvant for Vaccines with Different Immunological Requirements

Background It is now emerging that for vaccines against a range of diseases including influenza, malaria and HIV, the induction of a humoral response is insufficient and a substantial complementary cell-mediated immune response is necessary for adequate protection. Furthermore, for some diseases such as tuberculosis, a cellular response seems to be the sole effector mechanism required for protection. The development of new adjuvants capable of inducing highly complex immune responses with strong antigen-specific T-cell responses in addition to antibodies is therefore urgently needed. Methods and Findings Herein, we describe a cationic adjuvant formulation (CAF01) consisting of DDA as a delivery vehicle and synthetic mycobacterial cordfactor as immunomodulator. CAF01 primes strong and complex immune responses and using ovalbumin as a model vaccine antigen in mice, antigen specific cell-mediated- and humoral responses were obtained at a level clearly above a range of currently used adjuvants (Aluminium, monophosphoryl lipid A, CFA/IFA, Montanide). This response occurs through Toll-like receptor 2, 3, 4 and 7-independent pathways whereas the response is partly reduced in MyD88-deficient mice. In three animal models of diseases with markedly different immunological requirement; Mycobacterium tuberculosis (cell-mediated), Chlamydia trachomatis (cell-mediated/humoral) and malaria (humoral) immunization with CAF01-based vaccines elicited significant protective immunity against challenge. Conclusion CAF01 is potentially a suitable adjuvant for a wide range of diseases including targets requiring both CMI and humoral immune responses for protection.

Hypoxic response of Mycobacterium tuberculosis studied by metabolic labeling and proteome analysis of cellular and extracellular proteins.

The events involved in the establishment of a latent infection with Mycobacterium tuberculosis are not fully understood, but hypoxic conditions are generally believed to be the environment encountered by the pathogen in the central part of the granuloma. The present study was undertaken to provide insight into M. tuberculosis protein expression in in vitro latency models where oxygen is depleted. The response of M. tuberculosis to low-oxygen conditions was investigated in both cellular and extracellular proteins by metabolic labeling, two-dimensional electrophoresis, and protein signature peptide analysis by liquid chromatography-mass spectrometry. By peptide mass fingerprinting and immunodetection, five proteins more abundant under low-oxygen conditions were identified from several lysates of M. tuberculosis: Rv0569, Rv2031c (HspX), Rv2623, Rv2626c, and Rv3841 (BfrB). In M. tuberculosis culture filtrates, two additional proteins, Rv0363c (Fba) and Rv2780 (Ald), were found in increased amounts under oxygen limitation. These results extend our understanding of the hypoxic response in M. tuberculosis and potentially provide important insights into the physiology of the latent bacilli.

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.

Quality and Vaccine Efficacy of CD4+ T Cell Responses Directed to Dominant and Subdominant Epitopes in ESAT-6 from Mycobacterium tuberculosis

The ESAT-6 (early secretory antigenic target) molecule is a very important target for T cell recognition during infection with Mycobacterium tuberculosis. Although ESAT-6 contains numerous potential T cell epitopes, the immune response during infection is often focused toward a few immunodominant epitopes. By immunization with individual overlapping synthetic peptides in cationic liposomes (cationic adjuvant formulation, CAF01) we demonstrate that the ESAT-6 molecule contains several subdominant epitopes that are not recognized in H-2d/b mice either during tuberculosis infection or after immunization with ESAT-6/CAF01. Immunization with a truncated ESAT-6 molecule (Δ15ESAT-6) that lacks the immunodominant ESAT-61–15 epitope refocuses the response to include T cells directed to these subdominant epitopes. After aerosol infection of immunized mice, T cells directed to both dominant (ESAT-6-immunized) and subdominant epitopes (Δ15ESAT-6-immunized) proliferate and are recruited to the lung. The vaccine-promoted response consists mainly of double- (TNF-α and IL-2) or triple-positive (IFN-γ, TNF-α, and IL-2) polyfunctional T cells. This polyfunctional quality of the CD4+ T cell response is maintained unchanged even during the later stages of infection, whereas the naturally occurring infection stimulates a response to the ESAT-61–15 epitope that consist almost exclusively of CD4+ effector T cells. ESAT-6 and Δ15ESAT-6 both give significant protection against aerosol challenge with tuberculosis, but the most efficient protection against pulmonary infection is mediated by the subdominant T cell repertoire primed by Δ15ESAT-6.

Vaccine-Induced Th17 Cells Are Maintained Long-Term Postvaccination as a Distinct and Phenotypically Stable Memory Subset

ABSTRACT Th17 cells are increasingly being recognized as an important T helper subset for immune-mediated protection, especially against pathogens at mucosal ports of entry. In several cases, it would thus be highly relevant to induce Th17 memory by vaccination. Th17 cells are reported to exhibit high plasticity and may not stably maintain their differentiation program once induced, questioning the possibility of inducing durable Th17 memory. Accordingly, there is no consensus as to whether Th17 memory can be established unless influenced by continuous Th17 polarizing conditions. We have previously reported (T. Lindenstrøm, et al., J. Immunol. 182:8047–8055, 2009) that the cationic liposome adjuvant CAF01 can prime both Th1 and Th17 responses and promote robust, long-lived Th1 memory. Here, we demonstrate that subunit vaccination in mice with CAF01 leads to establishment of bona fide Th17 memory cells. Accordingly, Th17 memory cells exhibited lineage stability by retaining both phenotypic and functional properties for nearly 2 years. Antigen-specific, long-term Th17 memory cells were found to be mobilized from lung-draining lymph nodes to the lung following an aerosol challenge by Mycobacterium tuberculosis nearly 2 years after their induction and proliferated at levels comparable to those of Th1 memory cells. During the infection, the vaccine-induced Th17 memory cells expanded in the lungs and adapted Th1 characteristics, implying that they represent a metastable population which exhibits plasticity when exposed to prolonged Th1 polarizing, inflammatory conditions such as those found in the M. tuberculosis-infected lung. In the absence of overt inflammation, however, stable bona fide Th17 memory can indeed be induced by parenteral immunization.

Mapping Immune Reactivity toward Rv2653 and Rv2654: Two Novel Low-Molecular-Mass Antigens Found Specifically in the Mycobacterium tuberculosis Complex

New tools are urgently needed for the detection of latent tuberculosis (TB). We evaluated the diagnostic potential of 2 novel Mycobacterium tuberculosis complex-specific candidate antigens (Rv2653 and Rv2654) and investigated T cell recognition during natural infection in humans and experimental infection in guinea pigs. Peripheral blood mononuclear cells stimulated with peptide pools covering the full length of Rv2654 induced interferon- gamma release in 10 of 19 patients with TB. Neither Rv2654 single peptides nor Rv2654 pools were recognized by bacille Calmette-Guerin-vaccinated donors. However, peptides from Rv2653 were recognized by both patients group. The cross-reactive epitope(s) in Rv2653 were located in a 36-amino acid stretch in the center of the molecule. Rv2654 also induced M. tuberculosis-specific skin-test responses in 3 of 4 aerosol-infected guinea pigs. Rv2654 is a strongly recognized T cell antigen that is highly specific for TB and has potential as a novel cell-mediated immunity-based TB diagnostic agent.