BCG-Prime and boost with Esx-5 secretion system deletion mutant leads to better protection against clinical strains of Mycobacterium tuberculosis.

Although vaccination with BCG prevents disseminated forms of childhood tuberculosis (TB), it does not protect against pulmonary infection or Mycobacterium tuberculosis (Mtb) transmission. In this study, we generated a complete deletion mutant of the Mtb Esx-5 type VII secretion system (Mtb Δesx-5). Mtb Δesx-5 was highly attenuated and safe in immunocompromised mice. When tested as a vaccine candidate to boost BCG-primed immunity, Mtb Δesx-5 improved protection against highly virulent Mtb strains in the murine and guinea pig models of TB. Enhanced protection provided by heterologous BCG-prime plus Mtb Δesx-5 boost regimen was associated with increased pulmonary influx of central memory T cells (TCM), follicular helper T cells (TFH) and activated monocytes. Conversely, lower numbers of T cells expressing exhaustion markers were observed in vaccinated animals. Our results suggest that boosting BCG-primed immunity with Mtb Δesx-5 is a potential approach to improve protective immunity against Mtb. Further insight into the mechanism of action of this novel prime-boost approach is warranted.

Vaccination of guinea pigs with BCGΔBCG1419c transiently reduces hematogenous spread of M. tuberculosis to the spleen.

The guinea pig has proven to be a reliable model for testing vaccine candidates against tuberculosis (TB) because of its capacity to produce human-like disease associated to primary TB, thus providing a more stringent test of the ability of a vaccine to prevent disease and deaths. Here, the BCGΔBCG1419c vaccine candidate, which previously has been shown to provide protection in mice, was tested in a guinea pig model. We found that this vaccine candidate was as effective as parental BCG in reducing M. tuberculosis H37Rv replication in lungs, and significantly reducing hematogenous spread to spleen at 60 days post-infection in comparison with BCG. Moreover, lung histopathological examination revealed comparable protection between the parental and mutant BCG strains, with some differences in prevention of primary lesions or necrosis at a single time point post-infection in a strain-dependent manner. Our results show that the BCGΔBCG1419c vaccine candidate is as effective as BCG in reducing M. tuberculosis H37Rv replication in lungs and reducing lung pathology, as well as significantly improving control of its dissemination to spleens up to 60 days post-infection.

Biology of clinical strains of Mycobacterium tuberculosis with varying levels of transmission.

Transmission of Mycobacterium tuberculosis bacilli from one individual to another is the basis of the disease process. While considerable emphasis has been placed on the role of host mechanisms of resistance in establishing or preventing new infection, far less has been expended on understanding possible factors operative at the bacterial level. In this study we established a panel of clinical isolates of M. tuberculosis strains obtained from the Western Cape region of South Africa, each of which had been carefully tracked in terms of their degree of transmission in the community. Each of the panel were used to infect guinea pigs with 15-20 bacilli by aerosol exposure and the course of the infection then determined. Strains with different degrees of transmission could not be distinguished in terms of their capacity to grow in the main target organs of infected animals. However, rather surprisingly, while strains with no evidence of transmission [NOT] in general caused moderate to severe lung damage, this parameter in animals infected with highly transmitted [HT] strains was mostly mild. In terms of TH1 immunity these signals were strongest in these latter animals, as was IL-17 gene expression, whereas minimal signals for regulatory molecules including IL-10 and FoxP3 were seen across the entire panel. In terms of T cell numbers, responses of both CD4 and CD8 were both far faster and far higher in animals infected with the HT strains. At the gene expression level we observed a major three-fold difference [both up and down] between NOT and HT strains, but in terms of proteins of key interest only a few [including PD-L1 and HIF-3] showed major differences between the two groups. Overall, it was apparent that NOT strains were far more inflammatory that HT strains, and appeared to trigger a much larger number of genes, possibly explaining the observed damage to the lungs and progressive pathology. In contrast, the HT strains, while equally virulent, were more immunogenic and developed much stronger T cell responses, while keeping lung damage to a minimum. Hence, in terms of trying to explain the capacity of these strains to cause transmission, these results are clearly paradoxical.

Trying to See the Forest through the Trees: Deciphering the Nature of Memory Immunity to Mycobacterium tuberculosis.

The purpose of vaccination against tuberculosis and other diseases is to establish a heightened state of acquired specific resistance in which the memory immune response is capable of mediating an accelerated and magnified expression of protection to the pathogen when this is encountered at a later time. In the earliest studies in mice infected with Mycobacterium tuberculosis, memory immunity and the cells that express this were definable both in terms of kinetics of emergence, and soon thereafter by the levels of expression of markers including CD44, CD62L, and the chemokine receptor CCR7, allowing the identification of effector memory and central memory T cell subsets. Despite these initial advances in knowledge, more recent information has not revealed more clarity, but instead, has created a morass of complications-complications that, if not resolved, could harm correct vaccine design. Here, we discuss two central issues. The first is that we have always assumed that memory is induced in the same way, and consists of the same T cells, regardless of whether that immunity is generated by BCG vaccination, or by exposure to M. tuberculosis followed by effective chemotherapy. This assumption is almost certainly incorrect. Second, a myriad of additional memory subsets have now been described, such as resident, stem cell-like, tissue specific, among others, but as yet we know nothing about the relative importance of each, or whether if a new vaccine needs to induce all of these, or just some, to be fully effective.

GI-19007, a Novel Saccharomyces cerevisiae-Based Therapeutic Vaccine against Tuberculosis.

As yet, very few vaccine candidates with activity in animals against Mycobacterium tuberculosis infection have been tested as therapeutic postexposure vaccines. We recently described two pools of mycobacterial proteins with this activity, and here we describe further studies in which four of these proteins (Rv1738, Rv2032, Rv3130, and Rv3841) were generated as a fusion polypeptide and then delivered in a novel yeast-based platform (Tarmogen) which itself has immunostimulatory properties, including activation of Toll-like receptors. This platform can deliver antigens into both the class I and class II antigen presentation pathways and stimulate strong Th1 and Th17 responses. In mice this fusion vaccine, designated GI-19007, was immunogenic and elicited strong gamma interferon (IFN-γ) and interleukin-17 (IL-17) responses; despite this, they displayed minimal prophylactic activity in mice that were subsequently infected with a virulent clinical strain. In contrast, in a therapeutic model in the guinea pig, GI-19007 significantly reduced the lung bacterial load and reduced lung pathology, particularly in terms of secondary lesion development, while significantly improving survival in one-third of these animals. In further studies in which guinea pigs were vaccinated with BCG before challenge, therapeutic vaccination with GI-19007 initially improved survival versus that of animals given BCG alone, although this protective effect was gradually lost at around 400 days after challenge. Given its apparent ability to substantially limit bacterial dissemination within and from the lungs, GI-19007 potentially can be used to limit lung damage as well as facilitating chemotherapeutic regimens in infected individuals.

Mouse and Guinea Pig Models of Tuberculosis.

This article describes the nature of the host response to Mycobacterium tuberculosis in the mouse and guinea pig models of infection. It describes the great wealth of information obtained from the mouse model, reflecting the general availability of immunological reagents, as well as genetic manipulations of the mouse strains themselves. This has led to a good understanding of the nature of the T-cell response to the infection, as well as an appreciation of the complexity of the response involving multiple cytokine- and chemokine-mediated systems. As described here and elsewhere, we have a growing understanding of how multiple CD4-positive T-cell subsets are involved, including regulatory T cells, TH17 cells, as well as the subsequent emergence of effector and central memory T-cell subsets. While, in contrast, our understanding of the host response in the guinea pig model is less advanced, considerable strides have been made in the past decade in terms of defining the basis of the immune response, as well as a better understanding of the immunopathologic process. This model has long been the gold standard for vaccine testing, and more recently is being revisited as a model for testing new drug regimens (bedaquiline being the latest example).

Animal Models of Tuberculosis: An Overview.

This article provides an overview of the animal models currently used in tuberculosis research, both for understanding the basic science of the disease process and also for practical issues such as testing new vaccine candidates and evaluating the activity of potential new drugs. Animals range in size, from zebrafish to cattle, and in degrees of similarity to the human disease from both an immunological and pathologic perspective. These models have provided a great wealth of information (impossible to obtain simply from observing infected humans), but we emphasize here that one must use care in interpreting or applying this information, and indeed the true art of animal modeling is in deciding what is pertinent information and what might not be. These ideas are discussed in the context of current approaches in vaccine and drug development, including a discussion of certain limitations the field is currently facing in such studies.

Effect of bacillus Calmette-Guérin vaccination on CD4+Foxp3+ T cells during acquired immune response to Mycobacterium tuberculosis infection.

Increasing information has shown that many newly emerging strains of Mycobacterium tuberculosis, including the highly prevalent and troublesome Beijing family of strains, can potently induce the emergence of Foxp3(+)CD4 Tregs Although the significance of this is still not fully understood, we have previously provided evidence that the emergence of this population can significantly ablate the protective effect of BCG vaccination, causing progressive fatal disease in the mouse model. However, whether the purpose of this response is to control inflammation or to directly dampen the acquired immune response is still unclear. In the present study, we have shown, using both cell depletion and adoptive transfer strategies, that Tregs can have either properties. Cell depletion resulted in a rapid, but transient, decrease in the lung bacterial load, suggesting release or temporary re-expansion of effector immunity. Transfer of Tregs into Rag2(-/-)or marked congenic mice worsened the disease course and depressed cellular influx of effector T cells into the lungs. Tregs from infected donors seemed to preferentially depress the inflammatory response and granulocytic influx. In contrast, those from BCG-vaccinated and then challenged donors seemed more focused on depression of acquired immunity. These qualitative differences might be related to increasing knowledge reflecting the plasticity of the Treg response.

Vaccines to prevent tuberculosis infection rather than disease: Physiological and immunological aspects.

There is increasing enthusiasm and optimism that a vaccine could be developed that prevents infection rather than disease. In this article I discuss the fact that despite this optimism nothing has been produced so far that seems to have this capability, and moreover even the borderline between when infection ends and disease begins has not even been defined. To be effective such a vaccine, or at least the immunity it would generate, would have to work within the confines of the pulmonary physiological systems, which are complex. To date much of the emphasis here has turned away from T cell mediated immunity and towards establishing specific antibodies in the lungs. Here, I argue that with the exception of a possible exclusionary function, most claims of a protective role of antibody are completely over-blown. Finally, even if we had a potential "anti-infection" vaccine, how would we test and validate it?

The Efficacy of the BCG Vaccine against Newly Emerging Clinical Strains of Mycobacterium tuberculosis.

To date, most new vaccines against Mycobacterium tuberculosis, including new recombinant versions of the current BCG vaccine, have usually been screened against the laboratory strains H37Rv or Erdman. In this study we took advantage of our recent work in characterizing an increasingly large panel of newly emerging clinical isolates [from the United States or from the Western Cape region of South Africa], to determine to what extent vaccines would protect against these [mostly high virulence] strains. We show here that both BCG Pasteur and recombinant BCG Aeras-422 [used here as a good example of the new generation BCG vaccines] protected well in both mouse and guinea pig low dose aerosol infection models against the majority of clinical isolates tested. However, Aeras-422 was not effective in a long term survival assay compared to BCG Pasteur. Protection was very strongly expressed against all of the Western Cape strains tested, reinforcing our viewpoint that any attempt at boosting BCG would be very difficult to achieve statistically. This observation is discussed in the context of the growing argument made by others that the failure of a recent vaccine trial disqualifies the further use of animal models to predict vaccine efficacy. This viewpoint is in our opinion completely erroneous, and that it is the fitness of prevalent strains in the trial site area that is the centrally important factor, an issue that is not being addressed by the field.

Human IL-32 expression protects mice against a hypervirulent strain of Mycobacterium tuberculosis.

Silencing of interleukin-32 (IL-32) in a differentiated human promonocytic cell line impairs killing of Mycobacterium tuberculosis (MTB) but the role of IL-32 in vivo against MTB remains unknown. To study the effects of IL-32 in vivo, a transgenic mouse was generated in which the human IL-32γ gene is expressed using the surfactant protein C promoter (SPC-IL-32γTg). Wild-type and SPC-IL-32γTg mice were infected with a low-dose aerosol of a hypervirulent strain of MTB (W-Beijing HN878). At 30 and 60 d after infection, the transgenic mice had 66% and 85% fewer MTB in the lungs and 49% and 68% fewer MTB in the spleens, respectively; the transgenic mice also exhibited greater survival. Increased numbers of host-protective innate and adaptive immune cells were present in SPC-IL-32γTg mice, including tumor necrosis factor-alpha (TNFα) positive lung macrophages and dendritic cells, and IFN-gamma (IFNγ) and TNFα positive CD4(+) and CD8(+) T cells in the lungs and mediastinal lymph nodes. Alveolar macrophages from transgenic mice infected with MTB ex vivo had reduced bacterial burden and increased colocalization of green fluorescent protein-labeled MTB with lysosomes. Furthermore, mouse macrophages made to express IL-32γ but not the splice variant IL-32ß were better able to limit MTB growth than macrophages capable of producing both. The lungs of patients with tuberculosis showed increased IL-32 expression, particularly in macrophages of granulomas and airway epithelial cells but also B cells and T cells. We conclude that IL-32γ enhances host immunity to MTB.

The onset of adaptive immunity in the mouse model of tuberculosis and the factors that compromise its expression.

Mycobacterium tuberculosis (Mtb) has been evolving with its human host for over 50 000 years and is an exquisite manipulator of the human immune response. It induces both a strong inflammatory and a strong acquired immune response, and Mtb then actively regulates these responses to create an infectious lesion in the lung while maintaining a relatively ambulatory host. The CD4(+) T cell plays a critical yet contradictory role in this process by both controlling disseminated disease while promoting the development of the lesion in the lung that mediates transmission. In light of this manipulative relationship between Mtb and the human immune response, it is not surprising that our ability to vaccinate against tuberculosis (TB) has not been totally successful. To overcome the current impasse in vaccine development, we need to define the phenotype of CD4(+) T cells that mediate protection and to determine those bacterial and host factors that regulate the effective function of these cells. In this review, we describe the initiation and expression of T cells during TB as well as the fulminant inflammatory response that can compromise T-cell function and survival.

A new antibiotic with potent activity targets MscL.

The growing problem of antibiotic-resistant bacteria is a major threat to human health. Paradoxically, new antibiotic discovery is declining, with most of the recently approved antibiotics corresponding to new uses for old antibiotics or structurally similar derivatives of known antibiotics. We used an in silico approach to design a new class of nontoxic antimicrobials for the bacteria-specific mechanosensitive ion channel of large conductance, MscL. One antimicrobial of this class, compound 10, is effective against methicillin-resistant Staphylococcus aureus with no cytotoxicity in human cell lines at the therapeutic concentrations. As predicted from in silico modeling, we show that the mechanism of action of compound 10 is at least partly dependent on interactions with MscL. Moreover we show that compound 10 cured a methicillin-resistant S. aureus infection in the model nematode Caenorhabditis elegans. Our work shows that compound 10, and other drugs that target MscL, are potentially important therapeutics against antibiotic-resistant bacterial infections.

Differential Mycobacterium bovis BCG vaccine-derived efficacy in C3Heb/FeJ and C3H/HeOuJ mice exposed to a clinical strain of Mycobacterium tuberculosis.

The global epidemic caused by the bacterial pathogen Mycobacterium tuberculosis continues unabated. Moreover, the only available vaccine against tuberculosis, Mycobacterium bovis bacillus Calmette-Guérin (BCG), demonstrates variable efficacy. To respond to this global threat, new animal models that mimic the pathological disease process in humans are required for vaccine testing. One new model, susceptible C3Heb/FeJ mice, is similar to human tuberculosis in that these animals are capable of forming necrotic tubercle granulomas, in contrast to resistant C3H/HeOuJ mice. In this study, we evaluated the impact of prior BCG vaccination of C3Heb/FeJ and C3H/HeOuJ mice on exposure to a low-dose aerosol of Mycobacterium tuberculosis W-Beijing strain SA161. Both BCG-vaccinated murine strains demonstrated reduced bacterial loads 25 days after infection compared to controls, indicating vaccine efficacy. However, during chronic infection, vaccine efficacy waned in C3H/HeOuJ but not in C3Heb/FeJ mice. Protection in vaccinated C3Heb/FeJ mice was associated with reduced numbers of CD11b(+) Gr1(+) cells, increased numbers of effector and memory T cells, and an absence of necrotic granulomas. BCG vaccine efficacy waned in C3H/HeOuJ mice, as indicated by reduced expression of gamma interferon (IFN-γ) and increased expressions of interleukin-17 (IL-17), IL-10, and Foxp3 by T cells compared to C3Heb/FeJ mice. This is the first murine vaccine model system described to date that can be utilized to dissect differential vaccine-derived immune efficacy.

bis-Molybdopterin guanine dinucleotide is required for persistence of Mycobacterium tuberculosis in guinea pigs.

Mycobacterium tuberculosis is able to synthesize molybdopterin cofactor (MoCo), which is utilized by numerous enzymes that catalyze redox reactions in carbon, nitrogen, and sulfur metabolism. In bacteria, MoCo is further modified through the activity of a guanylyltransferase, MobA, which converts MoCo to bis-molybdopterin guanine dinucleotide (bis-MGD), a form of the cofactor that is required by the dimethylsulfoxide (DMSO) reductase family of enzymes, which includes the nitrate reductase NarGHI. In this study, the functionality of the mobA homolog in M. tuberculosis was confirmed by demonstrating the loss of assimilatory and respiratory nitrate reductase activity in a mobA deletion mutant. This mutant displayed no survival defects in human monocytes or mouse lungs but failed to persist in the lungs of guinea pigs. These results implicate one or more bis-MGD-dependent enzymes in the persistence of M. tuberculosis in guinea pig lungs and underscore the applicability of this animal model for assessing the role of molybdoenzymes in this pathogen.

The balance between protective and pathogenic immune responses in the TB-infected lung.

Tuberculosis is a disease of the lung, and efficient transmission is dependent on the generation of a lesion in the lung, which results in a bacterium-laden cough. Mycobacterium tuberculosis (Mtb) is able to manipulate both the innate and acquired immune response of the host. This manipulation results in an effective CD4(+) T cell response that limits disease throughout the body but can also promote the development of progressively destructive lesions in the lung. In this way Mtb infection can result in an ambulatory individual who has a lesion in the lung capable of transmitting Mtb. The inflammatory environment within the lung lesion is manipulated by Mtb throughout infection and can limit the expression of acquired immunity by a variety of pathways.

Tuberculosis vaccine types and timings.

Traditionally, the design of new vaccines directed against Mycobacterium tuberculosis, the most successful bacterial pathogen on the planet, has focused on prophylactic candidates that would be given to individuals while they are still young. It is becoming more apparent, however, that there are several types of vaccine candidates now under development that could be used under various conditions. Thus, in addition to prophylactic vaccines, such as recombinant Mycobacterium bovis BCG or BCG-boosting vaccines, other applications include vaccines that could prevent infection, vaccines that could be given in emergency situations as postexposure vaccines, vaccines that could be used to facilitate chemotherapy, and vaccines that could be used to reduce or prevent relapse and reactivation disease. These approaches are discussed here, including the type of immunity we are trying to specifically target, as well as the limitations of these approaches.

The formation of the granuloma in tuberculosis infection.

The development of the granuloma and its subsequent degeneration and necrosis, is the hallmark of infection caused by Mycobacterium tuberculosis. These structures probably evolved as primitive particle responses, but in mammals they are facilitated by the emerging acquired immune response, in which cytokines and chemokines help control their formation and integrity. In this brief review we discuss the pathology of these lesions in the two most widely used animal models (mice and guinea pigs). In addition, we argue against the idea that there is a balance between host immunity and bacterial survival, and that the latter possess mechanisms that control this, as some currently believe, and moreover discuss newer information regarding the ability of bacilli to persist in these structures long enough to eventually escape and become retransmitted.