Joseph P. Cassidy

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.

Control of latent Mycobacterium tuberculosis infection is dependent on CD8 T cells.

It is estimated that one‐third of the worlds population is infected with Mycobacterium tuberculosis, but that only 10% of infected people break down with the disease. In the remaining 90% the infection remains clinically latent. In the present study, the immune mechanisms controlling the latent phase of tuberculosis infection were evaluated in a mouse model of latency and reactivation. Mice aerosol‐infected with M. tuberculosis were treated with anti‐mycobacterial drugs resulting in very low, stable bacterial numbers (<500 CFU in the spleen and lung) for 10–12 weeks followed by reactivation of the disease with increasing bacterial numbers. During latency, pathological changes in the lung had almost completely resolved and lymphocyte number and turnover were at the pre‐infection level. The CD4 subset was highly active during the acute phase of infection and could be detected by intracellular staining for IFN‐γ as well as after antigen‐specific stimulation with mycobacterial antigens. The CD8 subset was not involved in the acute stage of infection, but this subset was active and produced IFN‐γ during the latent phase of infection. In vivo depletion of T cell subsets supported these findings with a 6–7‐fold increase in bacterial numbers in the lung following anti‐CD4 treatment during the acute phase, while anti‐CD8 treatment did not have an effect. The opposite was found during the latent phase where anti‐CD8 treatment as well as anti‐IFN‐γ treatment both resulted in a 10‐fold increase in bacterial numbers in the lung, while anti‐CD4 treatment induced only a modest change.

Early lesion formation in cattle experimentally infected with Mycobacterium bovis

Early lesion formation was examined in 13 calves inoculated intranasally with 2 x 10(7) colony-forming units of Mycobacterium bovis and killed either singly or in pairs at intervals of < or = 7 days from post-inoculation day (pid) 3 to pid 42. Immunological examinations were carried out before and after infection, and sequential necropsies were performed. M. bovis was recovered as early as pid 3, from the upper respiratory tract mucosae, retropharyngeal lymph nodes and caudal lung lobe. Gross tuberculous lesions were detected in both the upper respiratory tract mucosae and in the lungs of the calves killed from pid 14 onwards. Lesions were also present in the lymph nodes draining these areas. On histological examination, neutrophils appeared to play a key role in the earliest stages of lesion formation, and lesion mineralization was observed for the first time at pid 35. The contemporaneous development of lesions and cellular immunity, as demonstrated by in-vitro lymphocyte proliferation and interferon-gamma assay responses, provided further evidence of the role of immunopathogenic mechanisms in the development of bovine tuberculosis.

Modulation of Immune Responses to Mycobacterium bovis in Cattle Depleted of WC1+ γδ T Cells

ABSTRACT It is accepted that cell-mediated immune responses predominate in mycobacterial infections. Many studies have shown that CD4+ T cells produce Th1 cytokines, such as gamma interferon (IFN-γ), in response to mycobacterial antigens and that the cytolytic activity of CD8+ cells toward infected macrophages is important. However, the extent and manner in which γδ T cells participate in this response remain unclear. In ruminants, γδ T cells comprise a major proportion of the peripheral blood mononuclear cell population. We have previously shown that WC1+ γδ T cells are involved early in Mycobacterium bovis infection of cattle, but their specific functions are not well understood. Here we describe an in vivo model of bovine tuberculosis in which the WC1+ γδ T cells were depleted from the peripheral circulation and respiratory tract, by infusion of WC1+-specific monoclonal antibody, prior to infection. While no effects on disease pathology were observed in this experiment, results indicate that WC1+ γδ T cells, which become significantly activated (CD25+) in the circulation of control calves from 21 days postinfection, may play a role in modulating the developing immune response to M. bovis. WC1+-depleted animals exhibited decreased antigen-specific lymphocyte proliferative response, an increased antigen-specific production of interleukin-4, and a lack of specific immunoglobulin G2 antibody. This suggests that WC1+ γδ TCR+ cells contribute, either directly or indirectly, toward the Th1 bias of the immune response in bovine tuberculosis—a hypothesis supported by the decreased innate production of IFN-γ, which was observed in WC1+-depleted calves.

Specific Delayed-Type Hypersensitivity Responses to ESAT-6 Identify Tuberculosis-Infected Cattle

ABSTRACT Human and bovine tuberculosis have long been detected by skin testing with purified protein derivative (PPD), a complex mix of partly denatured mycobacterial antigens with suboptimal specificity. In the present study, skin tests based on ESAT-6, a recombinantly produced antigen highly specific for tuberculosis infection, were investigated. Although ESAT-6 was strongly recognized in vitro and induced high levels of gamma interferon, initial investigations demonstrated that higher doses of ESAT-6 than of PPD were needed to induce substantial delayed-type hypersensitivity reactions. Also, the kinetics of the skin test response differed for the two reagents; PPD showed maximal response at 72 h, but the response to ESAT-6 often peaked later at 96 h. Tests based on an optimized strategy (400 μg of ESAT-6 measured between 72 and 96 h), in cattle infected with Mycobacterium bovis (n = 22) and animals sensitized by exposure to environmental mycobacteria showed ESAT-6 to have a promising diagnostic potential (sensitivity, 82%; specificity, 100%; optimal cutoff, 3 mm), compared with PPD (sensitivity, 86%; specificity, 90%; optimal cutoff, 4 mm). Larger investigations are required to refine cutoff points for any new diagnostic test, but the present results indicate great potential for skin tests based on specific antigens for accurate in vivo diagnosis of tuberculosis.

Protective CD4 T Cells Targeting Cryptic Epitopes of Mycobacterium tuberculosis Resist Infection-Driven Terminal Differentiation

CD4 T cells are crucial to the control of Mycobacterium tuberculosis infection and are a key component of current vaccine strategies. Conversely, immune-mediated pathology drives disease, and recent evidence suggests that adaptive and innate responses are evolutionarily beneficial to M. tuberculosis. We compare the functionality of CD4 T cell responses mounted against dominant and cryptic epitopes of the M. tuberculosis 6-kDa early secreted Ag (ESAT-6) before and postinfection. Protective T cells against cryptic epitopes not targeted during natural infection were induced by vaccinating mice with a truncated ESAT-6 protein, lacking the dominant epitope. The ability to generate T cells that recognize multiple cryptic epitopes was MHC-haplotype dependent, including increased potential via heterologous MHC class II dimers. Before infection, cryptic epitope–specific T cells displayed enhanced proliferative capacity and delayed cytokine kinetics. After aerosol M. tuberculosis challenge, vaccine-elicited CD4 T cells expanded and recruited to the lung. In chronic infection, dominant epitope–specific T cells developed a terminal differentiated KLRG1+/PD-1lo surface phenotype that was significantly reduced in the cryptic epitope–specific T cell populations. Dominant epitope-specific T cells in vaccinated animals developed into IFN-γ– and IFN-γ,TNF-α–coproducing effector cells, characteristic of the endogenous response. In contrast, cryptic epitope–specific CD4 T cells maintained significantly greater IFN-γ+TNF-α+IL-2+ and TNF-α+IL-2+ memory-associated polyfunctionality and enhanced proliferative capacity. Vaccine-associated IL-17A production by cryptic CD4 T cells was also enhanced, but without increased neutrophilia/pathology. Direct comparison of dominant/cryptic epitope–specific CD4 T cells within covaccinated mice confirmed the superior ability of protective cryptic epitope–specific T cells to resist M. tuberculosis infection–driven T cell differentiation.

Adjuvant modulation of the cytokine balance in Mycobacterium tuberculosis subunit vaccines; immunity, pathology and protection

It is known that protection against tuberculosis is mediated primarily by T helper type 1 (Th1) cells but the influence of the Th1/Th2 balance of a vaccination response on the subsequent protection and pathology during infection has not been studied in detail. We designed a panel of Ag85B‐ESAT‐6 subunit vaccines based on adjuvants with different Th1/Th2‐promoting activities and studied cellular responses, bacterial replication and pathology in the lungs of mice infected with Mycobacterium tuberculosis. All vaccines induced cell‐mediated and humoral responses but with markedly different interferon‐γ : interleukin‐5 (IFN‐γ : IL‐5) and immunoglobulin G1 (IgG1) : IgG2 ratios. The vaccines promoted different levels of control of bacterial replication with the most efficient protection being exerted by cationic liposomes containing monophosphoryl lipid A and low to completely absent immunity with conventional aluminium. The level of protection correlated with the amount of IFN‐γ produced in response to the vaccine whereas there was no inverse correlation with the level of IL‐5. Characterizing a protective response was an accelerated recruitment of IL‐17 and IFN‐γ‐producing lymphocytes resulting in the early formation of granulomas containing clustered inducible nitric oxide synthase‐activated macrophages. In comparison, non‐protected mice exhibited a different inflammatory infiltrate rich in neutrophil granulocytes. This study indicates that the adjuvant component of a tuberculosis vaccine may be crucial in determining the kinetics by which effective granulomas, pivotal in controlling bacterial growth, are formed.

The detection of CD2+, CD4+, CD8+, and WC1+ T lymphocytes, B cells and macrophages in fixed and paraffin embedded bovine tissue using a range of antigen recovery and signal amplification techniques

In order to develop procedures to label the main bovine leucocyte populations in paraffin embedded sections, the immunoreactivity of 25 monoclonal antibodies (mAbs) to different leucocyte antigens was assessed with formal dichromate (FD5) and 10% formalin fixation, a battery of antigen retrieval (AR) methods, and the biotin-tyramide amplification system. All the leucocyte populations investigated (CD2+, CD4+, CD8+, WC1+ T lymphocytes, B cells and macrophages) were strongly and specifically detectable under an appropriate combination of mAb, AR method and signal amplification system. CD4 and CD8 required the most stringent conditions and could only be demonstrated in FD5 fixed sections. For detection of CD2, WC1+ T lymphocytes, B cells and macrophages, all the mAbs produced immunoreactivity in FD5 or formalin fixed tissues. The need to check a range of different AR methods is stressed, as the method of choice varied for each individual mAb. The incorporation of the signal amplification system was necessary to observe a strong signal and the complete distribution of CD4, CD8 and B cells. Fixation by FD5 proved to be better than formalin for the preservation of surface antigens but it was inferior for the detection of markers which were found to show cytoplasmic immunoreactivity, such as the macrophage marker MAC387 or the B cell markers BAQ155 or IL-A59.

ESAT-6 (EsxA) and TB10.4 (EsxH) Based Vaccines for Pre- and Post-Exposure Tuberculosis Vaccination

The ESX systems from Mycobacterium tuberculosis are responsible for the secretion of highly immunogenic proteins of key importance for bacterial survival and growth. The two prototypic proteins, ESAT-6 (EsxA from ESX-1) and TB10.4 (EsxH from ESX-3) share a lot of characteristics regarding genome organization, size, antigenic properties, and vaccine potential but the two molecules clearly have very different roles in bacterial physiology. To further investigate the role of ESAT-6 and TB10.4 as preventive and post-exposure tuberculosis vaccines, we evaluated four different fusion-protein vaccines; H1, H4, H56 and H28, that differ only in these two components. We found that all of these vaccines give rise to protection in a conventional prophylactic vaccination model. In contrast, only the ESAT-6-containing vaccines resulted in significant protection against reactivation, when administered post-exposure. This difference in post-exposure activity did not correlate with a difference in gene expression during infection or a differential magnitude or quality of the vaccine-specific CD4 T cells induced by ESAT-6 versus TB10.4-containing vaccines. The post-exposure effect of the ESAT-6 based vaccines was found to be influenced by the infectious load at the time-point of vaccination and was abolished in chronically infected animals with high bacterial loads at the onset of vaccination. Our data demonstrate that there are specific requirements for the immune system to target an already established tuberculosis infection and that ESAT-6 has a unique potential in post-exposure vaccination strategies.

Co-Infection of Cattle with Fasciola hepatica and Mycobacterium bovis - Immunological Consequences

Fasciola hepatica, the liver fluke, is a common parasite of cattle in much of the world. Previously, we have shown that cattle infected with F. hepatica have altered responsiveness (delayed type hypersensitivity reaction and cytokine responses) to M. bovis BCG infection. We hypothesized that co-infection with F. hepatica would, likewise, alter the immune response of cattle to virulent M. bovis infection, with possible implications for disease diagnosis and disease progression. Our previous work with F. hepatica/M. bovis BCG-infected cattle demonstrated a reduction in interferon (IFN)-gamma responsiveness in co-infected animals. Similar findings are reported here with virulent M. bovis following aerosol infection. The epidemiological significance of these findings, also, require exploration, particularly in view of the considerable resources devoted to the diagnosis and eradication of bovine tuberculosis, and the high prevalence of F. hepatica infection in areas where eradication has proved difficult.