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(Journal of Leukocyte Biology. 2001;70:1-10.)
© 2001 by Society for Leukocyte Biology

The search for new vaccines against tuberculosis

Ian M. Orme

Mycobacteria Research Laboratories, Department of Microbiology, Colorado State University, Fort Collins, Colorado

Correspondence: Ian Orme, Ph.D., Department of Microbiology, Colorado State University, Fort Collins CO 80523. E-mail: iorme{at}lamar.colostate.edu


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ABSTRACT
 
The failure of the BCG vaccine for tuberculosis in large, controlled clinical trials, coupled with the gradual consensus that it is mostly ineffective in preventing adult pulmonary disease in endemic areas, has led to a concerted effort to develop a new generation of vaccines. This work is ongoing in a variety of areas, including DNA vaccines, subunit vaccines, recombinant vaccines, and auxotrophic vaccines. Several such candidates are giving promising results in mouse and guinea pig, aerosol-challenge infection models and should move to clinical trials in the near future.

Key Words: BCG • animal models • T cells


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BACILLUS CALMETTE-GUERIN (BCG): THE CURRENT VACCINE
 
The BCG vaccine is named after the French scientists Calmette and Guerin who in 1908 isolated Mycobacterium bovis from a cow with tuberculous mastitis (a common symptom in cattle with bovine tuberculosis). They cultured this isolate in a glycerinated, beef/bile/potato medium, subculturing every 3 weeks, until 13 years and 230 subcultures later, the bacillus had lost all evidence of virulence [1 ].

After its acceptance as a vaccine for tuberculosis (TB) in humans, the BCG Pasteur Lille was widely distributed throughout the world to other institutions. As a result, many daughter substrains came into being, and recent molecular analysis has revealed numerous changes, gene deletions, as well as a suspicion that continued passage has attenuated BCG to almost complete avirulence [2 3 4 5 ].


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THE NEED FOR NEW VACCINES
 
Well over 1 billion people have been vaccinated with BCG since the first inoculation given in 1921. It can be given safely to children, it is inexpensive to produce, and side effects such as BCG adenitis are relatively minor.

As BCG became more widely accepted, various countries began to conduct controlled, clinical trials. At first, the news was good. For instance, in a trial in 1935 in native North Americans, there was an 82% reduction in deaths from TB [6 ]. Similar efficacy was seen in children in Chicago at high risk of disease [7 ] and in a study in the United Kingdom [8 ].

Other trials did not give such good results, however. Two trials in Georgia showed no protection after BCG, although in both, the incidence of disease was low (only a few cases) [1 ]. In contrast, in a study in a region of southern India, in which TB was prevalent, over 265,000 people were tested, and again the effects of BCG were zero [9 ].

This famous study began a process of complete re-evaluation of BCG as a vaccine. Although it is accepted that BCG does have some protective effect in children, particularly against forms such as meningeal TB, it does not prevent the emergence of pulmonary TB, particularly as the individual approaches adulthood [10 11 12 13 ].

Although there is a concerted effort ("directly observed therapy") to get drugs to the people who need them the most, as Kaufmann pointed out recently [14 ], an inexpensive vaccine that induces good immunity would be equally effective regardless of whether the infection was caused by drug-susceptible bacilli or by multiple drug resistance (MDR)-TB. In the long run, vaccination will be far more cost-effective than chemotherapy. Although not politically correct, there is a further caveat: namely, that large pharmaceutical companies probably dread the idea of finding a new, highly effective TB drug, given that (1) the cost of developing such drugs can be over $100 million, and (2) their marketing experts would remind them that the great majority of patients with active disease tend to be poor people, mostly in developing nations.


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WHAT SHOULD A NEW VACCINE DO?
 
Obviously, the best, new TB vaccine would prevent infection. Many of us think this is impossible, primarily because of the route of infection coupled with the intracellular nature of the organism. If it is not infection, then it is obviously disease; but which stage of the disease (Fig. 1 )?



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  		Figure 1. Target events for new TB vaccines. Although it is unlikely infection could be prevented, various stages of the disease process could be potentially targeted. The best, eventual outcome would be lesion sterilization, but if viable bacilli survive, then a competent, stable, long-lived, granulomatous response is needed to prevent subsequent reactivation disease.

Prevention of infection
Bacteria that are deposited within the alveolar region are probably taken up quickly by alveolar macrophages, and this is enhanced by binding a variety of factors including complement components, collectins, and pattern-recognition receptors [15 ].

It may well be impossible to block these mechanisms in any way, and so the immediate outcome resides on the ability of the macrophage to kill the bacillus. These cells flatten out across the alveolar surface and are tightly opposed; hence, if the bacillus cannot be killed, it begins to replicate and can potentially erode into the interstitium, thus creating an infectious site [16 ]. Because of this process, most scientists believe that a vaccine that can prevent infection is not possible.

Initial disease
A vaccine that can rapidly detect sites of infection and mobilize the immune response, thus preventing any further bacterial proliferation and potential dissemination, is the most practical likelihood. However, there may be physiologic limitations to this process. Even in mice highly immune to TB, the kinetics of influx of effector-memory T cells into the lungs after rechallenge are not especially rapid [17 ], and as a result, although about a tenfold reduction in the bacterial load can be observed, some bacterial proliferation seems inevitable.

To get memory cells to the site, gradients created by chemokines and other molecules that regulate local inflammation must be followed [18 ]. This takes time and, at least partially, involves aspects of the lung-innate response unaffected by vaccination. As we discuss elsewhere in this review, such multiple mechanisms may exist.

Post-exposure or immunotherapeutic vaccines
The concept here is to produce a vaccine that can be given to people with active disease, promoting increased clearance of the bacilli from the lungs.

The drawback is that even if an antigen-specific, T-cell pool could be induced, the location of the bacterial infection may preclude any beneficial effect. The core of the host response is to surround sites of infection with a granuloma response. These structures can grow large, and within them, only a small percentage of macrophages actually contain a viable bacillus [19 , 20 ]. Thus, not only will the T cell have to find these structures, it will also need the necessary integrins to burrow into the structure, find infected macrophages, and release interferon-{gamma} (IFN-{gamma}). This may indeed occur if the integrity of the granuloma is poor, and the structure is breaking down, but in situations where the granuloma is stable, it is hard to envisage how this could happen.

Vaccines against latency
I have expressed elsewhere my skepticism [21 ] that there actually is latent TB, and I do not think that there is an adequate animal model for this as yet, even if it does indeed exist. Semantics aside, everyone would probably agree that the scenario envisaged here—lesions that are not sterilized completely by host immunity or drug therapy—allows the survival of a few bacteria that have the potential to give rise to reactivation disease at a later time.

It is probable that if low-grade, chronic disease equates to latent disease, it is monitored by memory T cells in a sentinel or surveillance mode [22 ]. Thus, a good starting point would be to target this memory cell population in order to increase the pool size and also extend longevity. This strategy may not only involve CD4 cells; it is tempting to speculate that CD8 T cells, which we have shown recently to be scattered toward the outer edges of granulomas in the lung [23 ], are also performing such a role.


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WHAT ARE THE TARGET T-CELL POPULATIONS?
 
CD4 T cells
The central role of CD4 cells in immunity is well-established, beginning with the observation that this subset could transfer immunity to virulent infection [24 ] and more recently at the clinical level, with the knowledge that HIV-positive individuals with low CD4 T-cell counts are highly susceptible to TB [25 ].

Macrophages present antigens to CD4 T cells via class II major histocompatibility complex (MHC) molecules. They also secrete interleukin (IL)-1, stimulating IL-2 and IL-2 receptor (IL-2R) expression by the T cell. However, the key cytokine is IL-12. Mycobacterial infection is a potent inducer of this molecule from host macrophages, and it is pivotal in driving the T-helper cell type (Th1) response to TB [26 ].

Once committed to this pathway, the effector CD4 cell subset begins to secrete the key effector cytokine IFN-{gamma} [27 , 28 ]. This activates the infected macrophage to produce oxygen and nitrogen radicals and to assemble proton pumps on the phagosomal membrane, thus reducing the phagosomal pH [29 30 31 ].

The role of radicals in the containment of the infection is controversial. For some time, it was generally believed that oxygen radicals did not play any role, but more recent information using phox-knockout (KO) mice indicates a brief period during aerosol infection when lack of superoxide causes a transient rise in susceptibility [32 ]. Similarly, although blocking nitric oxide (NO) generation in intravenously (i.v.) infected mice causes a large increase in the bacterial load [33 , 34 ], the effects in the lungs of aerosol-infected mice are greatly reduced in models of M. tuberculosis and M. avium infection [35 , 36 ]. When taken in concert with the information [37 ] that clinical isolates of M. tuberculosis show a wide range of resistance to NO (some are completely oblivious), it must be concluded that the role of NO may be overstated.

Having said that, the role of NO may not be mycobactericidal but may be more in terms of regulation of the granuloma formation instead, as recent studies in NOS2-KO mice have suggested [35 , 36 ]. Perhaps this reflects its effects on local blood vessels.

Of course, the central target of the vaccine is CD4 memory T cells. To study these cells, the usual model is infection of mice followed by a prolonged period of chemotherapy to clear the bacilli [38 ]. Prior to rechallenge, these cells are small (low forward-scatter on flow analysis), resting T cells that express a CD44hi CD45RBlo cell-surface phenotype [39 ]. Following rechallenge, they mediate accelerated resistance to the infection rapidly [38 , 40 ].

Having said that, this is still an under-researched area, especially considering the pivotal importance of these cells to the ultimate goal of new TB vaccines. Their phenotype may not be stable, and a recent study suggests they may undergo reversion to a CD45RB+/hi phenotype [41 ]. Also, they may actually comprise two distinct subsets, based on their expression of the chemokine receptor CCR7 and their predilection to home to inflammatory sites within tissues or lymph nodes [42 ]. In my laboratory, this is an important issue, because I tend to rely on the CD44hi45lo phenotype to identify memory CD4 cells and use a footpad-challenge assay in which I measure cells with this phenotype accumulating in the draining, popliteal lymph node.

Perhaps the most important issue, however, is the longevity of the memory T-cell population. In fact, a gradual disappearance of these cells over time may be a major factor in unsuccessful BCG clinical trials. There is plenty of evidence now that memory T cells "turnover" from time to time, and this may be a homeostatic way of retaining these cells. Therefore, it is quite possible that if the stimuli for this process are too intense or too frequent, then this will have the opposite effect and diminish the memory T-cell pool.

Left unperturbed, the longevity of memory may be considerable. There is plenty of data indicating that people born in the first half of this century in first-world countries were skintest-positive and remain so to this day. (Presumably, this is mediated by CD4+ CCR7neg memory cells.) However, what if the individual is exposed to other stimuli constantly, such as environmental mycobacteria? In my laboratory, I have evidence that repeated exposure of BCG-vaccinated mice to dead M. avium gradually diminishes the size of the memory T-cell pool. To date, it is unknown exactly how this might occur; most of the literature describes studies in CD8 responses [43 44 45 46 ], and there, memory CD8 T-cell turnover can be driven nonspecifically by an IFN{alpha}ß/IL-15 axis. If a similar mechanism exists for CD4 memory cells, then it can be speculated that BCG vaccination may induce similar protection in everyone, but depending on where you live and to what you are exposed, it may directly influence the longevity of your memory T-cell population.

CD8 T cells
The role of CD8 T cells in immunity to TB remains elusive. Many years ago, I [24 ] was able to show that an enriched, CD8 T-cell population from mice immunized with a high dose of BCG prolonged the survival of irradiated mice exposed to an acute, high-dose, aerosol-challenge infection, and more recent work by Flynn and her colleagues [47 ] showed that ß2-microglobulin-deficient mice were highly susceptible to i.v. infection.

Using the same model, I have found that early resistance to aerosol infection does not seem directly attributable to lack of CD8 T cells, because direct comparison to CD8-KO mice indicates that these animals are not more susceptible until the lung infection is well into the chronic phase [48 , 49 ]. What causes the early rise in the lung bacterial load in ß2M-KO mice remains unknown, although class-1b-restricted natural killer (NK) cells or NKT cells might be candidates. Another molecule expressing ß2M, the CD1 molecule, is not involved, at least in the mouse model [48 ], although it seems to be important in human studies [50 , 51 ].

After i.v. infection, CD8 T cells accumulate in the lung lesions quite quickly [52 ]. We have seen a similar pattern after aerosol infection, but in our study [23 ], immunohistochemistry revealed that most CD8 cells tend to accumulate around blood vessels and bronchioles rather than infiltrate the lesions. Furthermore, this study also showed that during the chronic phase of the infection when the granulomas are large and stable, there are clear distinctions in distribution between CD4 and CD8 T cells, with the CD4 cells found in large aggregations within the structure and CD8 cells, more scattered with a tendency to be found toward the outside of the lesion (Fig. 2 ).



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  		Figure 2. Granulomatous structures forming 100 days after low-dose aerosol of mice with M. tuberculosis. Histochemical appearance using diaminobenzidine as the chromogen (brown) and hematoxylin as the counterstain. Consecutive sections stained for CD4 (A) and CD8 (B). Large numbers of CD4 are entering via a vessel (top right, A) and forming aggregates of lymphocytes throughout the epithelioid-macrophage field. In contrast, CD8 cells are lower in number, more scattered, found mostly as single cells, and tend to be distributed toward the periphery of the lesion. This may suggest a "sentinel" role for these latter cells. Original bar size, 100 µm. Photo courtesy of Dr. Oliver Turner._art>

Does this imply a sentinel role for the CD8 cells? Most of the viable bacilli are usually found toward the center of the granuloma, and this suggests that the role of the CD8 cells is to detect any dissemination of these bacilli to macrophages on the outside of this structure. This seems consistent with our observations in CD8-KO mice [48 , 49 ]; if this population is absent, bacteria gradually disseminate toward the outside of the lesion, with a concomitant rise in the lung bacterial load and death of the animal.

If this is the case, what are these cells doing, and what antigens are they recognizing? CD8 T cells could be activating the infected macrophages via secretion of IFN-{gamma}, or they could be killing them. This could be via direct lysis or by the delivery of apoptotic signals [49 ]. Viable bacilli could be then taken up by adjacent macrophages or even killed directly by granulysin [53 ].

Logically, apoptosis seems more likely. Lytic mechanisms would cause considerable, local tissue damage, and it should be remembered that cytolytic T lymphocyte (CTL) activity and bacterial damage by granulysin have only been demonstrated in vitro. In our study, mice lacking the perforin molecule look identical to controls during the chronic stage of the disease [49 ]. In the same study, we also observed that lack of CD95 (Fas) allowed regrowth of the infection during chronic disease, indicating that apoptotic mechanisms are indeed operative during this phase of the disease process.

Antigen presentation to CD8 T cells is restricted by class I MHC molecules, which requires processing of the antigen via a cytoplasmic rather than endosomal route. Therefore, it is possible that certain macrophages are able to control the infection well into the chronic-disease stage, but then this control is lost (maybe the cell is too far away from the aggregates of IFN-{gamma}-secreting CD4 cells in the granuloma, or their cytokine production has diminished). At this point, the bacilli grow unchecked in the cell, damaging the phagosome and allowing leakage into the cytoplasm. These are detected by the CD8 cells in the granuloma, which induce lysis or apoptosis in this cell, releasing the bacilli to be taken up by immunocompetent, adjacent macrophages.

If we wish to target these CD8 cells with a vaccine, we need to know what they are recognizing; as yet, this is unknown. Even then, would this be beneficial? If the end result is lysis, then amplifying this mechanism may actually worsen the lung pathology.


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INNATE MECHANISMS: COULD THEY BE TARGETED?
 
It takes a finite time for acquired immunity to become focused in the lung tissues, and yet mRNA for IFN-{gamma} can be detected before this event. Moreover, mice lacking CD4 cells or transgene mice that have no T-cell receptor (TCR) capable of recognizing mycobacterial antigens live much longer than IFN-{gamma}-KO mice (unpublished results). The explanation for these observations is that, in fact, the mouse has several, probably redundant, innate mechanisms of immunity in the lungs [15 ].

Yet again, it is accepted that these mechanisms are poorly understood. In humans, there is compelling evidence that CD3+4-8- T cells recognize mycobacterial lipoproteins presented by CD1 molecules [50 , 51 ]. Pattern-recognition Toll receptors on macrophages may also be involved [54 , 55 ], and this stimulation may be the source of IL-12 needed to drive early sources of IFN-{gamma}. NK or NKT cells may be one target of the IL-12, and indeed, these cells are known to be a potent source of IFN-{gamma} (Fig. 3 ).



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  		Figure 3. Possible innate mechanisms switched early on by mycobacterial infection.

There are other possibilities as well. A CD4 cell subset, which is also stimulated by mycobacterial lipoproteins, can be found in the lungs; this cell is also a strong source of IFN-{gamma} and differs from regular CD4 cells by lower expression of its TCR (unpublished results).

Finally, the role of {gamma}{delta} T cells remains elusive. We have suggested [16 ] that these play a regulatory role in cell trafficking rather than a directly protective role, given that {gamma}{delta}-KO mice have a similar bacterial load but a considerably different, much more pyogenic lung-tissue pathology [56 ]. It is interesting that {gamma}{delta} cells from humans seem to strongly recognize pyrophosphate-containing chemical moieties [57 58 59 60 61 62 ], whereas in animal models (mouse, cow), the targets seem to be proteins.

Would stimulation of these innate mechanisms be beneficial? It seems not. Not only do innate mechanisms lack any form of memory component, but also strong expression of innate immunity may completely preclude any proper emergence of acquired immunity. This would be acceptable if it could be ensured that innate immunity would completely sterilize the site of infection, but this seems unlikely to happen.


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THE FORGOTTEN LYMPHOCYTE: WHAT ABOUT B CELLS?
 
Despite the strong, classical literature to the contrary, the possibility that antibodies may play a protective role in TB has resurfaced [63 ]. In one recent study [64 ], bacilli were opsonized with a monoclonal antibody to arabinomannan (a component of the bacterial capsule—a contentious idea in itself) and then injected into mice. Whereas the control infection killed the animals, the mice given antibody-coated bacilli had considerable survival. However, whether this was a true, protective effect or a result of the fact that the opsonized bacteria were rafted together by the hundreds creating a classical particle response is open to debate.

However, should the role of B cells be discounted altogether? Perhaps not. B-cell KO mice show no changes in bacterial load after aerosol infection [65 ], but their lung granulomas are noticably smaller [66 ]. As we have shown recently [23 ], a simple explanation for this is that an appreciable number of lymphocytes within lung TB granulomas are indeed B cells. Why they are there is unknown; it may be a simple mistake because blood-vessel adhesion molecules at lesion sites would attract B-cell migration as well as T cells (the B cells think they are entering lymphoid tissue).

I would propose that the reason a role for B cells should not be completely discounted is because they may contribute to the continued stimulation of memory immunity. This is a contentious area; some argue that stimulation of memory cells is needed, and indeed, a recent study showed reduced T-cell memory in B-KO mice [67 ]. Other studies, such as those parking memory cells in MHC-deficient mice, argue the opposite [68 , 69 ].

If B cells are indeed needed, then it is believed that the antibody response to mycobacterial antigens results in deposition over time of immune complexes that may end up being captured by follicular, dendritic cells and periodically presented to passing memory T cells. If this is the case, then it can be experimentally tested to see if despite their capacity to deal with active disease, B-KO mice cannot generate persistent memory, measured as reduced resistance to rechallenge.


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BCG: IS IT GETTING A BAD RAP?
 
As discussed above, the bete noir of the field is the unavoidable fact that although BCG has been effective in certain controlled, clinical trials (with as much as 80% efficacy), in others, it has been disastrous, with no effect at all. Now, it is mostly accepted that although BCG does have positive effects in children, such as prevention of meningeal TB, it does not prevent pulmonary disease in adults.

An appreciation of this depends on a "glass half empty" or "glass half full" belief. Certainly, a percentage of scientists fall into the half-empty group, believing that BCG is essentially worthless and needs to be completely replaced. Conversely, the half-full membership points to the fact that in some of the trials, BCG did work well indeed, whereas in others it did not. So one question, still unanswered, is what is BCG doing when it does have a positive effect?

In fact, the stark reality is that this question has never been adequately answered. If the working hypothesis is believed, that BCG induces memory immunity, but this immunity is gradually lost, then perusal of the literature shows that recent, serious studies of this parameter in models can be counted on two hands [17 , 38 39 40 41 , 70 ]. The classical studies showing that BCG induces lymphocytes and that these can transfer resistance to naive recipients were done decades ago [71 , 72 ], long before there were CD markers and flow cytometers, knowledge of cytokines/chemokines barely existed, and no one had an inkling that there were CD1 molecules capable of presenting mycobacterial lipids and class-1b molecules.

In fact, it can be hypothesized that BCG can switch on a variety of mechanisms, as summarized in Figure 4 . Central to these is a potent, memory T-cell response [73 ]. In areas in which BCG is ineffective, or protection is of limited longevity, one possible explanation is that this memory T-cell pool is interfered with gradually or consumed, leading to an eventual loss of resistance and full susceptibility to disease by the teenage years. This may be a result of nonspecific stimulation of memory T-cell turnover and division, which may force a percentage of the pool into apoptosis, thus eventually reducing the pool size to a frequency whereby it is no longer effective. Although it is not yet understood how the size of the overall CD4 memory pool is regulated, it has been shown for CD8 memory cells that turnover can be induced via the production of IFN{alpha}ß and IL-15 after nonspecific activation of macrophages [46 , 74 ].



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  		Figure 4. BCG may be capable of switching on multiple, immune mechanisms. How many of these would have to be triggered by a new candidate vaccine still remains unanswered.


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IS REPLACING BCG THE BEST STRATEGY?
 
Several vaccine candidates give protection (i.e., reduced bacterial load 30 days post-exposure) similar to BCG and even on occasion, a little better [75 ]. However, it is evident that better values cannot be attained, not because of the intrinsic property of the vaccine itself, but because the small animal model has a limited "window of protection". In the mouse, this is quite small, with only 1.0–1.2 log protection usually achievable. In the guinea pig, in which the control inoculum grows to higher values because of the inherent susceptibility of this animal, a larger range of 2.0 to 3.0 logs can sometimes be attained (at least in the BCG controls) [76 , 77 ].

However, relying on "protection" alone may be limiting our options in screening potential vaccines. For example, we have made several observations whereby a particular test candidate does not induce a significant reduction in the day-30, bacterial-load assay. Such an example is the Ag85A-DNA vaccine, but if left alone, guinea pigs given this vaccine show excellent long-term survival [78 ]. Moreover, and perhaps much more importantly, the necrotic pathology that kills unprotected guinea pigs was replaced in the vaccinated pigs by a protective lymphocytic granuloma. Because a similar pathology occurs in human patients, this may be a positive effect, and this teaches us that a vaccine should not be completely discounted just because it cannot "beat BCG" in the 30-day assay.

It is also evident that testing a new vaccine against BCG is going to be difficult to achieve, simply because most children in areas of the world in which TB is endemic are given BCG as neonates. As a result, much of the experimental effort in my laboratory is to find ways to boost BCG as a vaccine.

This can be done in two ways. In the first method, the assumption is made that BCG is indeed effective (glass half-full), but memory immunity diminishes. If BCG-vaccinated mice are given BCG when young, their resistance to aerosol infection declines gradually as they approach old age. However, if given Ag85A protein-boosting in mid-life, their resistance to aerosol when elderly is equivalent to that of a young mouse [79 ].

A second way is to simply add a highly immunogenic vaccine to the BCG inoculum. In a recent study, guinea pigs were vaccinated with a mixture of BCG and an immunogenic fusion protein (M. tuberculosis 72f). Whereas the BCG, control guinea pigs began to die after about 300–350 days, four out of five pigs given the admixture remain alive (500 days, at the time of writing).


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TYPES OF NEW VACCINES
 
DNA vaccines
An exciting area of the vaccine field in general is the observation that microbial DNA sequences can be used as vaccine targets. As reviewed extensively elsewhere [80 ], the basic strategy is to identify an immunogenic protein, then isolate the gene and insert it into an expression plasmid that possesses a strong promotor gene, use this to transform bacterial cells so as to expand the plasmid, then isolate it, and use it as a vaccine. Once injected into animal-muscle cells, the plasmid is transcribed to RNA, and the cell expresses the protein.

This approach avoids the need for adjuvants, which are problematic in terms of other types of TB-vaccine candidates, and because a living mycobacterium is not involved, it can be safely put into an immunocompromised host. Once inoculated, experiments using a luciferase reporter gene suggest that the plasmid remains stable for some prolonged period of time, and the risk of genomic integration (a big worry) seems to be rare.

DNA vaccines seem to work well because they induce a type of pan-immune response; whatever the response of choice, DNA seems to induce them. After presentation by dendritic macrophages present in the muscle mass, a strong Th1 response can be observed, as well as strong CD8 CTL responses. (It is interesting that if given by "gene gun", which drives particles or liquid droplets into the dermis, Th2 responses are favored.) In addition, certain promotor systems, such as ubiquitin [81 ], may favor Th1 immunity and resulting protection in mice against TB infection.

Thus far, the majority of models tested have been viruses, but an increasing number of studies using bacterial, protozoal, and helminthic models are also appearing. In the TB field, several mycobacterial antigens have been targeted, including Ag85A, the phosphate-binding protein family PstS, the 36-kDa protein, ESAT-6, and the heat-shock proteins (hsp)60 and hsp70 [78 , 82 83 84 85 86 ].

The two most effective vaccines are against Ag85A and hsp60, although the results regarding the latter have unfortunately become controversial. In terms of the Ag85A vaccine, it reduces the day-30 bacterial load in mice. In guinea pigs, the load is appreciably not changed, but there is excellent, long-term survival, and the necrotic lung pathology seen in controls is completely prevented [78 ]. However, as a post-exposure vaccine in mice already infected with M. tuberculosis, the Ag85A DNA has no protective effect [87 ].

In mice given the hsp60 DNA (derived from M. leprae), the vaccine is equally, highly effective if given before or during TB infection and in both models causes a massive reduction in the bacterial load [84 ]. In addition, in a "Cornell style" mouse model in which the bacterial load is reduced to apparent sterility by chemotherapy, and then residual "latent" bacilli are reawakened by cortisol suppression of immunity, administration of three injections of the DNA vaccine totally prevented reactivation.

In our study, a DNA vaccine encoding the M. tuberculosis hsp60 gene gave different results [88 ]. The vaccine was completely unprotective in the mouse and guinea pig models, and in both cases, we noticed that many animals exhibited respiratory distress a few weeks after aerosol infection, requiring their euthanasia. Subsequent histologic analysis of the lungs of these animals revealed a severe necrotic, airway-inflammatory reaction; this was not seen in the vector controls or in animals vaccinated with Ag85 DNA. Given the close homology between the DNA vaccine genes involved, I cannot explain these divergent results as yet.

Subunit vaccines
Two studies published almost simultaneously in 1986 were the first to suggest that proteins secreted or expelled from the bacillus had the potential to be the key, protective antigens early during the host reponse [89 , 90 ], thus challenging the prevalent view at the time that constitutive proteins were the primary targets [91 , 92 ]. Gradually, this hypothesis has become widely accepted [93 ], and several laboratories were able to show protection in animal models using proteins from the culture-filtrate fraction soon thereafter [78 , 94 95 96 97 ].

This fraction contains many immunogenic proteins, some potent enough to induce some degree of immunity by themselves or as an admixture (Ag85 complex, ESAT-6, for example). However, in the more stringent guinea pig, none have been found as yet to be capable of inducing protection and long-term prevention of pathology seen in BCG controls.

Subunit vaccines need to be delivered in adjuvants, and it may be that the primary limitation is a failure to drive a sustained Th1 response rather than to find a truly effective protein candidate. Several adjuvants have been tried, and the most effective to date include monophosphoryl-lipid A (MPL), QS21 (a saponin), and dimethyl-dioctadecyl ammonium bromide (DDA).

This continues to be an active area of research. One recent advance is to identify immunogenic peptides or epitopes using cloned, human cell lines from PPD-positive individuals and then create fusion proteins using this information. Some of these fusion proteins are protective in the mouse and guinea pig models, and addition to a BCG vaccine as an admix significantly prolongs survival of guinea pigs when compared with BCG alone.

Another approach is to separate complex mixtures of proteins such as filtrates using isoelectric focusing and then in a second dimension, using gel electrophoresis; then electroelute each separated protein, and test whether they stimulate IFN-{gamma} secretion by T cells from infected mice. Those that are the most active are then identified by a proteomics approach using liquid chromatograph-mass spectrometry, and then predicted sequences are matched to known M. tuberculosis proteins using commercial software such as SEQUEST. Using this approach, several novel targets have been identified [98 ] and are now undergoing evaluations in animal models.

Auxotrophic vaccines
There are two concepts at work here. The first is that many people at risk of TB are also HIV-positive, and hence, even an attenuated bacterium such as BCG may cause fatal disease in these individuals. The second concept is to try to adapt the organism to the extent that it could itself be used as a vaccine, because the majority of people exposed to M. tuberculosis are actually able to express strong resistance to it.

To date, the major adaptation applied, in both cases, is auxotrophy. Several have been developed and tested now [99 , 100 ]. The initial results are encouraging in that protection can be conferred, and yet the vaccines themselves are cleared gradually, even in completely immunodeficient mice. If there is a drawback to this approach, it is the concern that the lack of persistence of the vaccine inoculum fails to adequately drive a memory T-cell response so that if the vaccine-challenge interval in the animal models is extended, protection measured tends to diminish. However, we are optimistic that this problem can be resolved.

Others
There are other categories of new vaccines being tested but with varying degrees of success. One such area is recombinant vaccines, which usually involve insertion of genes into the existing BCG vaccine. This has resulted in several innovative strategies [101 , 102 ], but when tested against BCG controls in mice, there is no evidence that the vaccine is more potent. In fairness, however, the point should be made that a true comparison has yet to be performed, because no one has attempted as yet to see if recombinant BCGs (rBCGs) could be better in long-term studies in guinea pigs, which might be a better form of evaluation of this class of vaccines.

In this regard, a recent study shows for the first time that protection in excess of that given by BCG can indeed be attained [103 ]. In that study, a BCG overexpressing the Ag85 protein was constructed, which gave about a half-log improvement in bacterial-load reduction compared with the BCG control in the guinea pig model. This is an exciting result, and it remains to be seen if this could result in better, long-term protection.

Another area of interest is to use avirulent mycobacteria as vaccines. There is some precedent for this; M. microti was tested in comparison with BCG in a trial in the United Kingdom [8 ], and protection was similar, although it should be noted that TB rates in children in the UK were already significantly falling during that time. There is nothing to suggest, however, that M. microti has any particular advantage over BCG and could be used to replace it.

A second candidate in this category is M. vaccae. In our animal models, M. vaccae was completely devoid of any immunological reactivity, but despite this, it has been seriously considered by some as a potential immunogen. At the end of the day, however, it did not fare well in a controlled, clinical trial (another validation that results in mice can predict results in humans) [104 ]. Having said that, one recent study did suggest some clinical improvement in patients receiving M. vaccae [105 ].


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VACCINE SAFETY TESTS
 
It is hoped that new vaccine candidates passing the tests in the animal models could then go to clinical evaluation. Unfortunately, life is not that simple because of a battery of further tests required by the FDA. These tests, which are described elsewhere in an excellent review [106 ], include, for example, a "general safety test" in which the vaccine is injected into animals to detect any possible toxicity problems; a "purity test", which includes a precise measure of the chemical composition of the vaccine; an "identity test", needed if the production site is also making other biologicals; a "sterility test", just to make sure there is no other bacterial contamination; a "freedom from mycobacteria test", to verify no contamination with live and potentially virulent M. tuberculosis organisms; and a "potency test", to verify no significant variations in biological activity from lot to lot.

Obviously, the purpose of these stringent requirements is to minimize the risk to the vaccine. However, as Brennan and his colleagues probably correctly argue [106 ], given the devastating problem that TB has become, one must accept that testing new vaccines in humans may indeed incur some risk.


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ACKNOWLEDGEMENTS
 
This work was supported by NIH grant AI-45707 and NIH Contract AI-75320. I am grateful to David McMurray, John Belisle, and Susan Baldwin for their contributions to some of the ideas expressed above.

Received January 9, 2001; revised March 1, 2001; accepted March 2, 2001.


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REFERENCES
 
    1
  1. Huebner, R. E. (1996) Bacillus of Calmette and Guerin (BCG) vaccine Rom, W. N. Garay, S. eds. Tuberculosis ,893-904 Little, Brown and Company New York.
    2. 2
  2. Oettinger, T., Jorgensen, M., Ladefoged, A., Haslov, K., Andersen, P. (1999) Development of the Mycobacterium bovis BCG vaccine: review of the historical and biochemical evidence for a genealogical tree Tuber. Lung Dis. 79,243-250[Medline]
    3. 3
  3. Behr, M. A., Small, P. M. (1997) Has BCG attenuated to impotence? Nature 389,133-134[Medline]
    4. 4
  4. Behr, M. A., Wilson, M. A., Gill, W. P., Salamon, H., Schoolnik, G. K., Rane, S., Small, P. M. (1999) Comparative genomics of BCG vaccines by whole-genome DNA microarray Science 284,1520-1523[Abstract/Free Full Text]
    5. 5
  5. Behr, M. A., Small, P. M. (1999) A historical and molecular phylogeny of BCG strains Vaccine 17,915-922[Medline]
    6. 6
  6. Aronson, J. D., Aronson, C. F., Taylor, H. C. (1958) A twenty-year appraisal of BCG vaccination in the control of tuberculosis Arch. Intern. Med. 101,881-893[Abstract/Free Full Text]
    7. 7
  7. Rosenthal, S. R., Loewinsohn, E., Graham, M., Liveright, D., Thorne, M., Johnson, V. (1961) BCG vaccination against tuberculosis in Chicago. A twenty-year study statistically analyzed Pediatrics 28,622-641[Abstract/Free Full Text]
    8. 8
  8. Hart, P. D., Sutherland, I. (1977) BCG and vole bacillus vaccines in the prevention of tuberculosis in adolescence and early adult life BMJ 2,293-295
    9. 9
  9. Tripathy, S. P. (1987) Fifteen-year follow-up of the Indian BCG prevention trial Bull. Int. Union Tuberc. Lung Dis. 62,69-72
    10. 10
  10. Colditz, G. A., Brewer, T. F., Berkey, C. S., Wilson, M. E., Burdick, E., Fineberg, H. V., Mosteller, F. (1994) Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature J. Am. Med. Assoc. 271,698-702[Abstract/Free Full Text]
    11. 11
  11. Rodrigues, L. C., Smith, P. G. (1990) Tuberculosis in developing countries and methods for its control Trans. R. Soc. Trop. Med. Hyg. 84,739-744[Medline]
    12. 12
  12. Rodrigues, L. C., Noel Gill, O., Smith, P. G. (1991) BCG vaccination in the first year of life protects children of Indian subcontinent ethnic origin against tuberculosis in England J Epidemiol. Community Health 45,78-80[Abstract/Free Full Text]
    13. 13
  13. Sterne, J. A., Rodrigues, L. C., Guedes, I. N. (1998) Does the efficacy of BCG decline with time since vaccination? Int. J. Tuberc. Lung Dis. 2,200-207[Medline]
    14. 14
  14. Kaufmann, S. H. (2000) Is the development of a new tuberculosis vaccine possible? Nat. Med. 6,955-960[Medline]
    15. 15
  15. Ferguson, J. S., Schlesinger, L. S. (2000) Pulmonary surfactant in innate immunity and the pathogenesis of tuberculosis Tuber. Lung Dis. 80,173-184[Medline]
    16. 16
  16. Orme, I. M., Cooper, A. M. (1999) Cytokine/chemokine cascades in immunity to tuberculosis Immunol. Today 20,307-312[Medline]
    17. 17
  17. Cooper, A. M., Callahan, J. E., Keen, M., Belisle, J. T., Orme, I. M. (1997) Expression of memory immunity in the lung following re-exposure to Mycobacterium tuberculosis Tuber. Lung Dis. 78,67-73[Medline]
    18. 18
  18. Rhoades, E. R., Cooper, A. M., Orme, I. M. (1995) Chemokine response in mice infected with Mycobacterium tuberculosis Infect. Immun. 63,3871-3877[Abstract]
    19. 19
  19. Rhoades, E. R., Frank, A. A., Orme, I. M. (1997) Progression of chronic pulmonary tuberculosis in mice aerogenically infected with virulent Mycobacterium tuberculosis Tuber. Lung Dis. 78,57-66[Medline]
    20. 20
  20. Orme, I. M. (1998) The immunopathogenesis of tuberculosis: a new working hypothesis Trends Microbiol 6,94-97[Medline]
    21. 21
  21. Orme, I. M. (2001) The latent tuberculosis bacillus: (I’ll let you know if I ever meet one) Int. J. Tuberc. Lung Dis. in press
    22. 22
  22. Orme, I. M., McMurray, D. N. (1995) The immune response to tuberculosis in animal models Rom, W. N. Garay, S. eds. Tuberculosis ,269-280 Little, Brown and Co. New York.
    23. 23
  23. Gonzalez-Juarerro, M., Turner, O. C., Orme, I. M. (2001) The temporal and spatial arrangement of lymphocytes within lung granulomas induced by aerosol infection with Mycobacterium tuberculosis Infect. Immun. in press
    24. 24
  24. Orme, I. M. (1987) The kinetics of emergence and loss of mediator T lymphocytes acquired in response to infection with Mycobacterium tuberculosis J. Immunol. 138,293-298[Abstract]
    25. 25
  25. Ackah, A. N., Coulibaly, D., Digbeu, H., Diallo, K., Vetter, K. M., Coulibaly, I. M., Greenberg, A. E., De Cock, K. M. (1995) Response to treatment, mortality, and CD4 lymphocyte counts in HIV-infected persons with tuberculosis in Abidjan, Cote d’Ivoire Lancet 345,607-610[Medline]
    26. 26
  26. Cooper, A. M., Magram, J., Ferrante, J., Orme, I. M. (1997) Interleukin 12 (IL-12) is crucial to the development of protective immunity in mice intravenously infected with mycobacterium tuberculosis J. Exp. Med. 186,39-45[Abstract/Free Full Text]
    27. 27
  27. Cooper, A. M., Dalton, D. K., Stewart, T. A., Griffin, J. P., Russell, D. G., Orme, I. M. (1993) Disseminated tuberculosis in interferon gamma gene-disrupted mice J. Exp. Med. 178,2243-2247[Abstract/Free Full Text]
    28. 28
  28. Flynn, J. L., Chan, J., Triebold, K. J., Dalton, D. K., Stewart, T. A., Bloom, B. R. (1993) An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection J. Exp. Med. 178,2249-2254[Abstract/Free Full Text]
    29. 29
  29. Sturgill-Koszycki, S., Schlesinger, P. H., Chakraborty, P., Haddix, P. L., Collins, H. L., Fok, A. K., Allen, R. D., Gluck, S. L., Heuser, J., Russell, D. G. (1994) Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase Science 263,678-681[Abstract/Free Full Text]
    30. 30
  30. Russell, D. G. (1995) Of microbes and macrophages: entry, survival and persistence Curr. Opin. Immunol. 7,479-484[Medline]
    31. 31
  31. Russell, D. G., Sturgill-Koszycki, S., Vanheyningen, T., Collins, H., Schaible, U. E. (1997) Why intracellular parasitism need not be a degrading experience for Mycobacterium Philos. Trans. R. Soc. Lond. B Biol. Sci. 352,1303-1310[Abstract/Free Full Text]
    32. 32
  32. Cooper, A. M., Segal, B. H., Frank, A. A., Holland, S. M., Orme, I. M. (2000) Transient loss of resistance to pulmonary tuberculosis in p47(phox-/-) mice Infect. Immun. 68,1231-1234[Abstract/Free Full Text]
    33. 33
  33. Chan, J., Xing, Y., Magliozzo, R. S., Bloom, B. R. (1992) Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages J. Exp. Med. 175,1111-1122[Abstract/Free Full Text]
    34. 34
  34. Chan, J., Tanaka, K., Carroll, D., Flynn, J., Bloom, B. R. (1995) Effects of nitric oxide synthase inhibitors on murine infection with Mycobacterium tuberculosis Infect. Immun. 63,736-740[Abstract]
    35. 35
  35. Cooper, A. M., Pearl, J. E., Brooks, J. V., Ehlers, S., Orme, I. M. (2000) Expression of the nitric oxide synthase 2 gene is not essential for early control of mycobacterium tuberculosis in the murine lung Infect. Immun. 68,6879-6882[Abstract/Free Full Text]
    36. 36
  36. Ehlers, S., Kutsch, S., Benini, J., Cooper, A., Hahn, C., Gerdes, J., Orme, I., Martin, C., Rietschel, E. T. (1999) NOS2-derived nitric oxide regulates the size, quantity and quality of granuloma formation in Mycobacterium avium-infected mice without affecting bacterial loads Immunology 98,313-323[Medline]
    37. 37
  37. Rhoades, E. R., Orme, I. M. (1997) Susceptibility of a panel of virulent strains of Mycobacterium tuberculosis to reactive nitrogen intermediates Infect. Immun. 65,1189-1195[Abstract]
    38. 38
  38. Orme, I. M. (1988) Characteristics and specificity of acquired immunologic memory to Mycobacterium tuberculosis infection J. Immunol. 140,3589-3593[Abstract]
    39. 39
  39. Griffin, J. P., Orme, I. M. (1994) Evolution of CD4 T-cell subsets following infection of naive and memory immune mice with Mycobacterium tuberculosis Infect. Immun. 62,1683-1690[Abstract/Free Full Text]
    40. 40
  40. Andersen, P., Heron, I. (1993) Specificity of a protective memory immune response against Mycobacterium tuberculosis Infect. Immun. 61,844-851[Abstract/Free Full Text]
    41. 41
  41. Andersen, P., Smedegaard, B. (2000) CD4(+) T-cell subsets that mediate immunological memory to Mycobacterium tuberculosis infection in mice Infect. Immun. 68,621-629[Abstract/Free Full Text]
    42. 42
  42. Sallusto, F., Lenig, D., Forster, R., Lipp, M., Lanzavecchia, A. (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions Nature 401,708-712[Medline]
    43. 43
  43. Sprent, J., Tough, D. F. (1994) Lymphocyte life-span and memory Science 265,1395-1400[Abstract/Free Full Text]
    44. 44
  44. Sprent, J., Tough, D. F., Sun, S. (1997) Factors controlling the turnover of T memory cells Immunol. Rev. 156,79-85[Medline]
    45. 45
  45. Tough, D. F., Sprent, J. (1994) Turnover of naive- and memory-phenotype T cells J. Exp. Med. 179,1127-1135[Abstract/Free Full Text]
    46. 46
  46. Tough, D. F., Sun, S., Zhang, X., Sprent, J. (2000) Stimulation of memory T cells by cytokines Vaccine 18,1642-1648[Medline]
    47. 47
  47. Flynn, J. L., Goldstein, M. M., Triebold, K. J., Koller, B., Bloom, B. R. (1992) Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection Proc. Natl. Acad. Sci. USA 89,12013-12017[Abstract/Free Full Text]
    48. 48
  48. D’Souza, C. D., Cooper, A. M., Frank, A. A., Ehlers, S., Turner, J., Bendelac, A., Orme, I. M. (2000) A novel nonclassic beta2-microglobulin-restricted mechanism influencing early lymphocyte accumulation and subsequent resistance to tuberculosis in the lung Am. J. Respir. Cell Mol. Biol. 23,188-193[Abstract/Free Full Text]
    49. 49
  49. Turner, J., D’Souza, C. D., Pearl, J. E., Marietta, P., Noel, M., Frank, A. A., Appelberg, R., Orme, I. M., Cooper, A. M. (2001) CD8- and CD95/95L-dependent mechanisms of resistance in mice with chronic pulmonary tuberculosis Am. J. Respir. Cell Mol. Biol. 24,203-209[Abstract/Free Full Text]
    50. 50
  50. Bendelac, A. (1995) CD1: presenting unusual antigens to unusual T lymphocytes Science 269,185-186[Free Full Text]
    51. 51
  51. Porcelli, S. A., Segelke, B. W., Sugita, M., Wilson, I. A., Brenner, M. B. (1998) The CD1 family of lipid antigen-presenting molecules Immunol. Today 19,362-368[Medline]
    52. 52
  52. Serbina, N. V., Liu, C. C., Scanga, C. A., Flynn, J. L. (2000) CD8+ CTL from lungs of Mycobacterium tuberculosis-infected mice express perforin in vivo and lyse infected macrophages J. Immunol. 165,353-363[Abstract/Free Full Text]
    53. 53
  53. Stenger, S., Mazzaccaro, R. J., Uyemura, K., Cho, S., Barnes, P. F., Rosat, J. P., Sette, A., Brenner, M. B., Porcelli, S. A., Bloom, B. R., Modlin, R. L. (1997) Differential effects of cytolytic T cell subsets on intracellular infection Science 276,1684-1687[Abstract/Free Full Text]
    54. 54
  54. Brightbill, H. D., Libraty, D. H., Krutzik, S. R., Yang, R. B., Belisle, J. T., Bleharski, J. R., Maitland, M., Norgard, M. V., Plevy, S. E., Smale, S. T., Brennan, P. J., Bloom, B. R., Godowski, P. J., Modlin, R. L. (1999) Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors Science 285,732-736[Abstract/Free Full Text]
    55. 55
  55. Thoma-Uszynski, S., Kiertscher, S. M., Ochoa, M. T., Bouis, D. A., Norgard, M. V., Miyake, K., Godowski, P. J., Roth, M. D., Modlin, R. L. (2000) Activation of toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10 J. Immunol. 165,3804-3810[Abstract/Free Full Text]
    56. 56
  56. D’Souza, C. D., Cooper, A. M., Frank, A. A., Mazzaccaro, R. J., Bloom, B. R., Orme, I. M. (1997) An anti-inflammatory role for gamma delta T lymphocytes in acquired immunity to Mycobacterium tuberculosis J. Immunol. 158,1217-1221[Abstract]
    57. 57
  57. Burk, M. R., Mori, L., De Libero, G. (1995) Human V gamma 9-V delta 2 cells are stimulated in a cross-reactive fashion by a variety of phosphorylated metabolites Eur. J. Immunol. 25,2052-2058[Medline]
    58. 58
  58. Constant, P., Davodeau, F., Peyrat, M. A., Poquet, Y., Puzo, G., Bonneville, M., Fournie, J. J. (1994) Stimulation of human gamma delta T cells by nonpeptidic mycobacterial ligands Science 264,267-270[Abstract/Free Full Text]
    59. 59
  59. Kabelitz, D., Bender, A., Schondelmaier, S., Schoel, B., Kaufmann, S. H. (1990) A large fraction of human peripheral blood gamma/delta + T cells is activated by Mycobacterium tuberculosis but not by its 65-kD heat shock protein J. Exp. Med. 171,667-679[Abstract/Free Full Text]
    60. 60
  60. Lang, F., Peyrat, M. A., Constant, P., Davodeau, F., David-Ameline, J., Poquet, Y., Vie, H., Fournie, J. J., Bonneville, M. (1995) Early activation of human V gamma 9V delta 2 T cell broad cytotoxicity and TNF production by nonpeptidic mycobacterial ligands J. Immunol. 154,5986-5994[Abstract]
    61. 61
  61. Pfeffer, K., Schoel, B., Gulle, H., Kaufmann, S. H., Wagner, H. (1990) Primary responses of human T cells to mycobacteria: a frequent set of gamma/delta T cells are stimulated by protease-resistant ligands Eur. J. Immunol. 20,1175-1179[Medline]
    62. 62
  62. Tanaka, Y., Morita, C. T., Nieves, E., Brenner, M. B., Bloom, B. R. (1995) Natural and synthetic non-peptide antigens recognized by human gamma delta T cells Nature 375,155-158[Medline]
    63. 63
  63. Glatman-Freedman, A., Casadevall, A. (1998) Serum therapy for tuberculosis revisited: reappraisal of the role of antibody-mediated immunity against Mycobacterium tuberculosis Clin. Microbiol. Rev. 11,514-532[Abstract/Free Full Text]
    64. 64
  64. Teitelbaum, R., Glatman-Freedman, A., Chen, B., Robbins, J. B., Unanue, E., Casadevall, A., Bloom, B. R. (1998) A mAb recognizing a surface antigen of Mycobacterium tuberculosis enhances host survival Proc. Natl. Acad. Sci. USA 95,15688-15693[Abstract/Free Full Text]
    65. 65
  65. Johnson, C. M., Cooper, A. M., Frank, A. A., Bonorino, C. B., Wysoki, L. J., Orme, I. M. (1997) Mycobacterium tuberculosis aerogenic rechallenge infections in B cell-deficient mice Tuber. Lung Dis. 78,257-261[Medline]
    66. 66
  66. Bosio, C. M., Gardner, D., Elkins, K. L. (2000) Infection of B cell-deficient mice with CDC 1551, a clinical isolate of Mycobacterium tuberculosis: delay in dissemination and development of lung pathology J. Immunol. 164,6417-6425[Abstract/Free Full Text]
    67. 67
  67. Linton, P-J., Harbertson, J., Bradley, L. M. (2000) A critical role for B cells in the development of memory CD4 cells J. Immunol. 165,5558-5565[Abstract/Free Full Text]
    68. 68
  68. Murali-Krishna, K., Lau, L. L., Sambhara, S., Lemonnier, F., Altman, J., Ahmed, R. (1999) Persistence of memory CD8 T cells in MHC class I-deficient mice Science 286,1377-1381[Abstract/Free Full Text]
    69. 69
  69. Swain, S. L., Hu, H., Huston, G. (1999) Class II-independent generation of CD4 memory T cells from effectors Science 286,1381-1383[Abstract/Free Full Text]
    70. 70
  70. Andersen, P., Andersen, A. B., Sorensen, A. L., Nagai, S. (1995) Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice J. Immunol. 154,3359-3372[Abstract]
    71. 71
  71. Kostiala, A. A., Lefford, M. J., McGregor, D. D. (1978) Immunological memory in tuberculosis. 2. Mediators of protective immunity, delayed hypersensitivity and macrophage migration inhibition in central lymph Cell. Immunol. 41,9-19[Medline]
    72. 72
  72. Lefford, M. J., McGregor, D. D. (1974) Immunological memory in tuberculosis. I. Influence of persisting viable organisms Cell. Immunol. 14,417-428[Medline]
    73. 73
  73. Orme, I. M. (1988) Evidence for a biphasic memory T-cell response to high dose BCG vaccination in mice Tubercle 69,125-131[Medline]
    74. 74
  74. Zhang, X., Sun, S., Hwang, I., Tough, D. F., Sprent, J. (1998) Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15 Immunity 8,591-599[Medline]
    75. 75
  75. Orme, I. M. (1999) Vaccination against tuberculosis: recent progress Adv. Vet. Med. 41,135-143[Medline]
    76. 76
  76. McMurray, D. N. (1994) Guinea pig model of tuberculosis Bloom, B. R. eds. Tuberculosis: Pathogenesis, Protection, and Control ,135-147 ASM Washington, DC.
    77. 77
  77. McMurray, D. N., Dai, G., Phalen, S. (1999) Mechanisms of vaccine-induced resistance in a guinea pig model of pulmonary tuberculosis Tuber. Lung Dis. 79,261-266[Medline]
    78. 78
  78. Baldwin, S. L., D’Souza, C., Roberts, A. D., Kelly, B. P., Frank, A. A., Lui, M. A., Ulmer, J. B., Huygen, K., McMurray, D. M., Orme, I. M. (1998) Evaluation of new vaccines in the mouse and guinea pig model of tuberculosis Infect. Immun. 66,2951-2959[Abstract/Free Full Text]
    79. 79
  79. Brooks, J. V., Frank, A. A., Keen, M., Belisle, J. T., Orme, I. M. (2001) A boosting vaccine for tuberculosis Infect. Immun. 69,2714-2717[Abstract/Free Full Text]
    80. 80
  80. Huygen, K. (1998) DNA vaccines: application to tuberculosis Int. J. Tuberc. Lung Dis. 2,971-978[Medline]
    81. 81
  81. Delogu, G., Howard, A., Collins, F. M., Morris, S. L. (2000) DNA vaccination against tuberculosis: expression of a ubiquitin-conjugated tuberculosis protein enhances antimycobacterial immunity Infect. Immun. 68,3097-3102[Abstract/Free Full Text]
    82. 82
  82. Huygen, K., Content, J., Denis, O., Montgomery, D. L., Yawman, A. M., Deck, R. R., DeWitt, C. M., Orme, I. M., Baldwin, S., D’Souza, C., Drowart, A., Lozes, E., Vandenbussche, P., Van Vooren, J. P., Liu, M. A., Ulmer, J. B. (1996) Immunogenicity and protective efficacy of a tuberculosis DNA vaccine Nat. Med. 2,893-898[Medline]
    83. 83
  83. Lowrie, D. B., Silva, C. L., Colston, M. J., Ragno, S., Tascon, R. E. (1997) Protection against tuberculosis by a plasmid DNA vaccine Vaccine 15,834-838[Medline]
    84. 84
  84. Lowrie, D. B., Tascon, R. E., Bonato, V. L., Lima, V. M., Faccioli, L. H., Stavropoulos, E., Colston, M. J., Hewinson, R. G., Moelling, K., Silva, C. L. (1999) Therapy of tuberculosis in mice by DNA vaccination Nature 400,269-271[Medline]
    85. 85
  85. Tanghe, A., Lefevre, P., Denis, O., D’Souza, S., Braibant, M., Lozes, E., Singh, M., Montgomery, D., Content, J., Huygen, K. (1999) Immunogenicity and protective efficacy of tuberculosis DNA vaccines encoding putative phosphate transport receptors J. Immunol. 162,1113-1119[Abstract/Free Full Text]
    86. 86
  86. Tascon, R. E., Colston, M. J., Ragno, S., Stavropoulos, E., Gregory, D., Lowrie, D. B. (1996) Vaccination against tuberculosis by DNA injection Nat. Med. 2,888-892[Medline]
    87. 87
  87. Turner, J., Rhoades, E. R., Keen, M., Belisle, J. T., Frank, A. A., Orme, I. M. (2000) Effective pre-exposure tuberculosis vaccines fail to protect when given in an immunotherapeutic mode Infect. Immun. 68,1706-1709[Abstract/Free Full Text]
    88. 88
  88. Turner, O. C., Roberts, A. D., Frank, A. A., Phalen, S. W., McMurray, D. M., Content, J., Denis, O., D’Souza, S., Tanghe, A., Huygen, K., Orme, I. M. (2000) Lack of protection in mice and necrotizing bronchointerstitial pneumonia with bronchiolitis in guinea pigs immunized with vaccines directed against the hsp60 molecule of Mycobacterium tuberculosis Infect. Immun. 68,3674-3679[Abstract/Free Full Text]
    89. 89
  89. Orme, I. M., Collins, F. M. (1986) Crossprotection against nontuberculous mycobacterial infections by Mycobacterium tuberculosis memory immune T lymphocytes J. Exp. Med. 163,203-208[Abstract/Free Full Text]
    90. 90
  90. Rook, G. A., Steele, J., Barnass, S., Mace, J., Stanford, J. L. (1986) Responsiveness to live M. tuberculosis, and common antigens, of sonicate-stimulated T cell lines from normal donors Clin. Exp. Immunol. 63,105-110[Medline]
    91. 91
  91. Kaufmann, S. H., Vath, U., Thole, J. E., Van Embden, J. D., Emmrich, F. (1987) Enumeration of T cells reactive with Mycobacterium tuberculosis organisms and specific for the recombinant mycobacterial 64-kDa protein Eur. J. Immunol. 17,351-357[Medline]
    92. 92
  92. Young, R. A. (1990) Stress proteins and immunology Annu. Rev. Immunol. 8,401-420[Medline]
    93. 93
  93. Orme, I. M., Andersen, P., Boom, W. H. (1993) T cell response to Mycobacterium tuberculosis J. Infect. Dis. 167,1481-1497[Medline]
    94. 94
  94. Andersen, P. (1994) Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins Infect. Immun. 62,2536-2544[Abstract/Free Full Text]
    95. 95
  95. Horwitz, M. A., Lee, B. W., Dillon, B. J., Harth, G. (1995) Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis Proc. Natl. Acad. Sci. USA 92,1530-1534[Abstract/Free Full Text]
    96. 96
  96. Pal, P. G., Horwitz, M. A. (1992) Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis Infect. Immun. 60,4781-4792[Abstract/Free Full Text]
    97. 97
  97. Roberts, A. D., Sonnenberg, M. G., Ordway, D. J., Furney, S. K., Brennan, P. J., Belisle, J. T., Orme, I. M. (1995) Characteristics of protective immunity engendered by vaccination of mice with purified culture filtrate protein antigens of Mycobacterium tuberculosis Immunology 85,502-508[Medline]
    98. 98
  98. Covert, B. A., Spencer, J. S., Orme, I. M., Belisle, J. T. (2001) The application of proteomics in defining the T cell antigens of Mycobacterium tuberculosis Proteomics 1,574-586[Medline]
    99. 99
  99. Chambers, M. A., Williams, A., Gavier-Widen, D., Whelan, A., Hall, G., Marsh, P. D., Bloom, B. R., Jacobs, W. R., Hewinson, R. G. (2000) Identification of a Mycobacterium bovis BCG auxotrophic mutant that protects guinea pigs against M. bovis and hematogenous spread of Mycobacterium tuberculosis without sensitization to tuberculin Infect. Immun. 68,7094-7099[Abstract/Free Full Text]
    100. 100
  100. Hondalus, M. K., Bardarov, S., Russell, R., Chan, J., Jacobs, W. R., Jr, Bloom, B. R. (2000) Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis Infect. Immun. 68,2888-2898[Abstract/Free Full Text]
    101. 101
  101. Murray, P. J., Aldovini, A., Young, R. A. (1996) Manipulation and potentiation of antimycobacterial immunity using recombinant bacille Calmette-Guerin strains that secrete cytokines Proc. Natl. Acad. Sci. USA 93,934-939[Abstract/Free Full Text]
    102. 102
  102. O’Donnell, M. A., Aldovini, A., Duda, R. B., Yang, H., Szilvasi, A., Young, R. A., DeWolf, W. C. (1994) Recombinant Mycobacterium bovis BCG secreting functional interleukin-2 enhances gamma interferon production by splenocytes Infect. Immun. 62,2508-2514[Abstract/Free Full Text]
    103. 103
  103. Horwitz, M. A., Harth, G., Dillon, B. J., Maslesa-Galic, S. (2000) Recombinant bacillus Calmette-Guerin (BCG) vaccines expressing the mycobacterium tuberculosis 30-kDa major secretory protein induce greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model Proc. Natl. Acad. Sci. USA 97,13853-13858[Abstract/Free Full Text]
    104. 104
  104. . No authors listed (1999) Immunotherapy with Mycobacterium vaccae in patients with newly diagnosed pulmonary tuberculosis: a randomised controlled trial. Durban Immunotherapy Trial Group Lancet 354,116-119[Medline]
    105. 105
  105. Johnson, J. L., Kamya, R. M., Okwera, A., Loughlin, A. M., Nyole, S., Hom, D. L., Wallis, R. S., Hirsch, C. S., Wolski, K., Foulds, J., Mugerwa, R. D., Ellner, J. J. (2000) Randomized controlled trial of Mycobacterium vaccae immunotherapy in non-human immunodeficiency virus-infected Ugandan adults with newly diagnosed pulmonary tuberculosis. The Uganda-Case Western Reserve University research collaboration J. Infect. Dis. 181,1304-1312[Medline]
    106. 106
  106. Brennan, M. J., Collins, F. M., Morris, S. L. (1999) Propelling novel vaccines directed against tuberculosis through the regulatory process Tuber. Lung Dis. 79,145-151[Medline]



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