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I thought I would just remark that these conferences are in the name of Sir Mark Oliphant. When I was a high school student, Sir Mark Oliphant, after he had retired, actually came to our Speech Day, and I remember noting this man and the enthusiasm with which he came and spoke about science. It is an honour to be speaking at a conference named after him.
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Today I am talking about vaccine developments against tuberculosis. Tuberculosis is really one of the world’s most effective parasites of humans. The reason is that, although when it infects humans a small proportion, up to 5 per cent, progress to disease, nevertheless 90 per cent of people live successfully with the tuberculosis organism in their lungs, and only 5 per cent of people with latent TB – and in Australia an excellent study done by Guy Marks in refugees in Liverpool showed that in Liverpool it was 7.3 per cent of Vietnamese refugees – reactivated over a prolonged period of time. So this is a very successful organism at living with the host. And that is one of the problems in creating more effective vaccines.
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For that reason, in fact, this organism infects a third of humanity. That results in a huge case load of tuberculosis each year, and during the current decades 35 million more deaths. Unfortunately, infection by this successful parasite is now being driven by another very successful organism which we have just been hearing about, HIV infection, and also by the emergence of drug resistance.
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Over the last 15 years, unfortunately, there has been an increase throughout the world of tuberculosis.
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That has been driven by drug resistance – and the most worrying strains of drug resistant organisms, those identified two years ago in South Africa, which were resistant to up to five drugs, have now been identified in four different continents and 28 different countries, so tuberculosis really is capable of rapid spread.
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And it has also been driven by HIV infection.
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There is an unfortunate correlation between the frequency of HIV infection in the community and the reactivation of tuberculosis.
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That has led to the Stop TB Partnership of WHO, and it is recognised now that new preventive tools, both in terms of vaccines and in terms of preventive therapy, are urgently required.
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Now, what kind of vaccine do we have? BCG is one of those historical vaccines which developed through attenuation – over a 13-year period, so greater than the length of an NHMRC grant – but this vaccine was only formally tested properly from the 1950s onwards, and it really protects effectively against neonatal tuberculosis. There is an interesting property, demonstrated in Guinea-Bissau, that those who had received BCG vaccine had a 2 per cent reduction in mortality against other infections over the next 12 months of life. So BCG and the immunity it induces have effects in childhood beyond the control of tuberculosis.
But it is more problematic in adults, and particularly it is more problematic in preventing pulmonary tuberculosis. In those studies where it has protected – and there are a number of those – the protection wanes after 10 to 15 years.
So what we really need for tuberculosis is a more effective vaccine to protect against pulmonary tuberculosis, and a vaccine which has greater durability.
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Over the last 10 to 15 years there have been considerable efforts directed towards this. There have been three broad approaches.
One approach, which has been greatly assisted by the sequencing of the TB and many other mycobacterial genomes, has been the development of new attenuated strains. In our own lab we have tested such an attenuated strain. We have shown that it is more protective than BCG, but unfortunately it was also more virulent in immunodeficient mice. So the prospects for an attenuated strain of tuberculosis I think are somewhat limited.
The second approach is to develop improved BCG vaccine strains, and that is what I would like to discuss today.
And the third approach is to use a viable vector, possibly delivered either as DNA protein or a viral vector, but a non-mycobacterial viable vector which can deliver appropriate proteins of M. tuberculosis and induce immunity, particularly to boost the waning effects of BCG safely.
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Firstly we will consider BCG strains. BCG was derived from Mycobacterium bovis, and there are in fact 16 areas of the virulent Mycobacterium bovis genome which are deleted in BCG. But not all BCG is the same. In fact, the original strain which was distributed from France to many other serum laboratories around the world has undergone subsequent attenuation. So different strains of BCG are lacking different members of these regions of deletion.
The father of all deletions is RD1, and deletion of the RD1 strain, which contains 13 open reading frames, reduces the virulence of Mycobacterium bovis. But, as you can see from this slide, there are up to 129 genes, and a number of these are secreted proteins which are very immunogenic. So the concept has been whether one could use, particularly, secreted proteins which were deleted from BCG, perhaps as subunit vaccines – or, in this case, we have engineered BCG to express a fusion protein of a dominant gene present in BCG and TB plus ESAT-6, which is a gene deleted in the RD1 region.
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This strain is very immunogenic, and it is a little more protective. Here it is shown that, following an aerosol challenge with tuberculosis, the recombinant strain of BCG (in red) is about half a log more protective in the lung and a little more protective in the spleen. We have subsequently done experiments with guinea pigs and shown that this does increase the survival, but it doesn’t increase the survival against virulent tuberculosis to a great extent. So we need to have a more potent vaccine than this.
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In more recent years we have been considering how to engineer and utilise BCG, engineering it with components of the host immune system. We have tested about half a dozen different strains of BCG over-expressing different murine cytokines, with a view to use in humans.
The one we have found most effective is actually a strain of BCG over-expressing GM-CSF. GM-CSF is a critical molecule in the induction of immunity, in its role of the generation and activation of dendritic cells and, through dendritic cells, the activation of Th1 T cells. It also has a role in the expansion of macrophages, and in fact a GM-CSF deficiency actually leads to macrophage defects in the lung. And GM-CSF can also activate macrophages, and have an effector role.
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This is a strain of GM-CSF, and one would be a little surprised that in fact this mammalian cytokine can be expressed by a prokaryote, BCG. Somehow or other it gets out of BCG. We engineer it so that it has a mycobacterial secretory signal sequence. And then it gets out of the phagolysosome, out of the macrophage, and is in fact effective. At the top centre here is functional, showing the presence of BCG-derived GM-CSF, and when we take that we can expand dendritic cells (shown in blue) and when we use this BCG:GM-CSF to infect dendritic cells, we get a greatly enhanced IL-12 response.
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Anthony Ryan, a PhD student in the laboratory, immunised mice with this BCG:GM-CSF strain, and showed (in red on the slide) that we developed a stronger antigen-specific interferon-gamma T cell response, and that this T cell response persisted up to day 56 and longer.
You will note, even in this experiment in the draining lymph node cells (DLNs), how the T cell response to BCG starts to wane, even as early as day 56.
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When we then infected these mice with tuberculosis, we found consistently, in multiple experiments, that we increased the protective effect in the spleen but we didn’t increase the protective effect in the lung. There is a kind of secret known to mycobacteriologists that, in experimental tuberculosis, BCG and live organisms always protect better in the spleen. They reduce dissemination from the lung to other organs. And interestingly it is disseminated tuberculosis in childhood for which BCG is particularly effective.
However, we know that to increase the protective effect of BCG in the human population we need to reduce pulmonary tuberculosis.
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So then Jamie Triccas, Anthony Ryan and Jonathan Nambiar have been immunising mice intranasally with recombinant BCG expressing GM-CSF. Shown in blue on the slide, we have just the measurement of GM-CSF protein, measured 24 hours after infection in these mice. You can see that in the cervical and the mediastinal lymph nodes and the lung, one can detect GM-CSF.
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This is associated with the increased expansion of dendritic cells in the animal, in the lung, and also increased dendritic cells in the draining lymph nodes. That is shown here in red, where there is increased frequency of CD11c+ dendritic cells at day 7 and at day 28, in these mice.
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Then when we look at the effect of the recruitment of T cells into the lungs of these animals, we see that this increases the recruitment of both CD4 T cells (shown here in blue) and CD8 T cells, into the lung, as compared to BCG alone.
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This, importantly, is associated with the expansion of interferon-gamma secreting T cells, both in the mediastinal lymph nodes and in the lungs of these animals.
So we have then been interested to see whether this increased interferon-gamma T cell response translates into increased protective immunity.
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When these mice are given aerosol tuberculosis, there is in fact a very significant enhancement in the protective immune response in the lung, as well as a reduction in dissemination to the spleen. You will note that this was done four weeks after the immunisation, and Jonathan Nambiar has subsequently extended this out to three months, which is the time for a longer time point used in the mouse model, and this increased protective effect is maintained.
Interestingly, the increased protective effect is demonstrated by a marked reduction in the immunopathology in the lungs. For our colleagues who work with cattle, trying to protect against bovine tuberculosis, their model of protection in cattle is, in fact, the reduction of the number of tubercules in the lung, and this correlates in this model.
So we have here a version of BCG which definitely increases the protective effect in the lung. Could we ever utilise this in humans?
Recently some chemical engineers in Harvard developed a new technique for drying BCG without freezing it. This dramatically increases the viability of BCG and it reduces the clumping of the organisms. They have demonstrated that you can deliver this as an aerosol to animals, with a view to using aerosol delivery in humans. So we are now currently collaborating with the Aeras Foundation and putting human GM-CSF into a strain of BCG which is being used in vaccine trials.
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The other approach which I would like to discuss, just briefly, is the idea of making better DNA vaccines by using only a single subunit. Certainly, as recently as 15 years ago it was thought to be impossible to protect against chronic intracellular infection with tuberculosis by using a dead vaccine. The reason is that if you take BCG, heat it and immunise an animal, it loses its protective effect. So it was thought that one could not immunise with a dead vaccine.
This field was actually changed by DNA vaccination, where it became possible to test relatively easily a large number of different candidate genes. There are 3956 genes in TB, and you will notice that there is a large number of secreted proteins. Even though tuberculosis is apparently an indolent organism, it is manipulating its environment. In fact, it gets its energy source by secreting enzymes which metabolise host membranes and provide energy.
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So, if you take individual candidate genes from these secreted proteins, and put them into a DNA vaccine, you can protect against tuberculosis. A large number of different genes have been tested in this way, and there are well over 20 different candidate genes, or in some cases proteins, which have been shown to protect against tuberculosis.
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That is exemplified here by work from a student in our lab, Arun Kamath. Some time ago he showed that if you increased the number of DNA vaccines, up to in this case three, you can develop increased protective efficacy against virulent tuberculosis infection.
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We have now shown this with proteins, but I show this recent work from Nick West, in our lab, to highlight a particular point. Here we have taken a family of secreted proteins – the particular proteins don’t matter – and you can see that by taking recombinant protein in a potent adjuvant we develop a very strong Th1 T cell response, a very healthy T cell response. But in fact only one of these, CLP1, is significantly protective. Other antigens which are just as immunogenic are not as protective in this model. That is just to highlight that the actual correlates of protective immunity we are using at the moment are suboptimal.
However, with this particular antigen, which we can give individually or as a fusion, we can show significant lung protection.
But the second point about this slide is that BCG is better than the individual protein in terms of the protective efficacy. And that is almost always the case. Despite one or two claims to the contrary, almost all subunit vaccines against TB are not as effective as BCG. It is remarkable that one protein induces protective immunity, and perhaps that protein could be used as a boost for BCG, but as a primary vaccine currently it is suboptimal
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For that reason, we have been looking at ways of enhancing the immune response to individual subunit vaccines. The technique we have been using is testing different cytokines as DNA vaccines – in this case delivering interleukin-12 or interleukin-23 – at the same time as we immunise with DNA-85B. You will see here that we get enhanced T cell recognition and very significantly enhanced interferon-gamma T cell responses, both in CD4 and in CD8 components.
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When we use this strategy, we can increase the protective immunity, in this case to DNA-85B, significantly.
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We have tested quite a range of cytokines, and this is just to highlight, using a different cytokine, IL-27, which is a heterodimeric cytokine, that early in immune responses it expands Th1 T cells but late in chronic immune responses dampens down Th1 T cell responses. IL-27 actually inhibits IL-12 induced increase in interferon-gamma T cell responses, and in fact on the right-hand side here you can see the increased protective efficacy with IL-12 and IL-23 – these are not cumulative, it seems to be a similar effect – whereas IL-27 itself does not increase protection. In fact, it reverses protection, and it reverses the increased protective effect of interleukin-12.
This is just to highlight the complexity of the cellular immune response to this organism, and that as yet we still have a way to go to understand the basic biology, as well as using that information to enhance the protective effect of vaccines. But it is possible, using a combination of, in this case, a DNA vaccine and interleukin-12, to get protective immunity in the lung equivalent to that provided by BCG.
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So where are we now in applying this? Well, one of the themes of the use of subunit vaccines is to use them to boost the effects of BCG. As yet the data on their effectiveness is mixed. There have only been six studies reported in the literature; two of them have shown increased benefit in guinea pigs and three of them have shown increased benefit in mice. In our own hands, and I know in the hands of others, we have also found boosting techniques which don’t work.
However, it is encouraging that, certainly with the MVA85, you can increase the survival of guinea pigs given BCG. And there is now one study in humans, from the Oxford group, where they have shown that the delivery of MVA85 to humans immunised with BCG, from one to 38 years earlier, has shown enhancement of the Th1 T cell response.
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The planned use of these vaccines is now starting to be rolled out, and there is a plan to complete the testing of a number of these candidates by 2015. Listed here are five candidates which are currently in either phase I or phase II human trials. There are actually better candidates than these, coming behind them. So I think that for improving immunisation against tuberculosis, although it is very difficult, there is a realistic prospect that it may be achieved.
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Discussion
Question: I wonder what you think about BCG in the context of HIV, since that’s one of the more complex problems we have to face as vaccinologists.
Warwick Britton: I think it has been widely recognised that in HIV-endemic countries, boosting with live vaccines is not feasible. And BCG will continue to be used in children, because it undoubtedly has an effect of increasing protection. So definitely any boosting vaccine to be used in adolescents or adults, which is the target audience, will need to be of a non-viable nature.
For many years it was thought that BCG could be given safely to infants in HIV-endemic countries, without infecting HIV-infected subjects, but recently it has been clear that HIV-infected children who are given BCG have an increased risk of BCGosis. But then it becomes a difficult question, and I think the current view is that we should persist with the use of BCG because of the benefit to the vast majority of children. In situations where you can screen the mother and know that the mother has been screened for HIV it is simple. And if the mother is positive, then BCG is not given, until it has been shown the child is not infected.
If the situation is, as in many countries, that screening for HIV is still not occurring, BCG immunisation is continuing. But I guess it highlights the benefit to the population and the risk to the individual.
Question: I have a question a little bit peripheral to the theme of your talk. I hadn’t actually realised that so many antigens of mycobacteria in general were secreted proteins and presumably get outside the infected cell, which renders it even more difficult to understand why it is so difficult to show antibodies against many of these antigens. I know that with Johne’s disease, in the veterinary world, you can have gram quantities of bacteria, but if you try and find some antibody in those infected cattle it is pretty tough. Are these things completely non-immunogenic? Do they get out of the infected cell? And why do we not see more antibody to secreted proteins?
Warwick Britton: Well, I think they get out of the infected bacterium into the phagolysosome. In fact, most of them are involved in the assembly of the cell wall. What differentiates mycobacteria from other species is the complexity of their cell wall, and that is assembled. And the majority of these are involved in the assembly of the cell wall. You can show the more abundant ones are actually present in the phagosome, and you can show that even exosomes from infected macrophages contain, particularly, lipoproteins.
The other major function of these secreted proteins is either to recruit energy – so the group of antigens I showed you are lipases, involved in breaking down cell membranes – but also there is a number now that have been shown to be essential in blocking phagosome maturation. So they secrete, for instance, serine/threonine kinase, which is active on host cells, and acid phosphatase, active in stripping phosphate. So they actually manipulate the internal environment of the phagosome to maximise their survival.
Question (cont.): And most are sequestered within the cell?
Warwick Britton: Yes.
Question: So there is evidence that prior exposure to bacterial antigens in the lung and the resulting increase in T cells into the lung is associated with increased susceptibility to secondary infections, to something like influenza. I was wondering if you have ever tested your vaccinated mice to see if your BCG:GM-CSF vaccine might increase or decrease susceptibility to something like influenza.
Warwick Britton: No, we haven’t, but in fact we are planning to do that with some colleagues in Melbourne, exactly for the reasons you highlight. There is quite an old literature that TB can influence influenza infection, and vice versa, but it is mainly based on animal experiments of 30 or 40 years ago. There isn’t recent data, but we are interested in that very issue.
Question: Warwick, in your recombinant protein immunogenicity studies, it is interesting that you got some that are equally immunogenic but vary in protection. Have you tried mixing some of the weakly protective ones to see whether you are getting a subtlety in the immune responses, so that the combination and total immune response may be protective whereas individual responses are not?
Warwick Britton: No, we haven’t. We have tested various combinations to find what is the optimal combination, but we haven’t found an individual protein that suppresses. There are carbohydrate molecules in mycobacteria that do suppress host immunity, and there is a lipoprotein which suppresses class II processing, but in terms of actually acting as, I guess, alternative or inhibitory antigens, no, we haven’t done that.
Question: You showed a very nice protocol for inducing protection against TB by a combination of GM-CSF and BCG. But I am just wondering: it may be acceptable for the uninfected host cases, that GM-CSF is only effective in uninfected host macrophages and APCs. I am a tumour neurologist. If the GM-CSF were injected into the right tumour-bearing host with the tumour antigen, that is where GM-CSF [inaudible] suppress and actively suppress macrophages and DCs. And so GM-CSF is only effective in the [inaudible] host, and the [inaudible] DC. So I think if you tried with the HIV infected children to induce a protective immunity against TB, if you used the GM-CSF and BCG protocol is [inaudible]. What do you think about that?
Warwick Britton: We realise it is complex. One of the Holy Grails of this area is to come up with a vaccine which can increase protection in prior infected people – in other words, to take that 90 per cent of people who have got latent infection and to have a vaccination which can reduce their chance of reactivation. That is a very difficult thing to test. There are some models emerging in mice to do that, and we need to bear in mind, I guess, the point that you are raising. But it has been shown that if you have a vaccine that you can use not only to prevent infection but also to increase the capacity for clearance of the organism in prior infected individuals, that would return the most benefit in terms of control of tuberculosis in the long run.
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