References

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Andersen P 1994 Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins. Infect Immun 62:2536—2544 Baldwin S, D'Souza C, Roberts A et al 1998 Evaluation of neew vaccines in the mouse and guinea pig model of tuberculosis. Infect Immun, in press Barry MA, Lai WC, Johnston SA 1995 Protection against mycoplasma infection using expression-library immunization. Nature 377:632—635 Denis O, Tanghe A, Palfliet K et al 1998 Vaccination with plasmid DNA encoding Ag85A stimulates a broader CD4+ and CD8+ T cell epitopic repertoire than infection with M. tuberculosis H37Rv. Infect Immun 66:1527—1533 Donnelly JJ, Ulmer JB, Shiver JW, Liu MA 1997 DNA vaccines. Annu Rev Immunol 15:617— 648

Guleria I, Teitelbaum R, McAdam RA, Kalpana G, Jacobs WR, Bloom BR 1996 Auxotrophic vaccines for tuberculosis. Nat Med 2:334—336 Huygen K, Content J, Denis O et al 1996 Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat Med 2:893—898

Lim EM, Rauzier J, Timm J et al 1995 Identification of mycobacterial tuberculosis DNA

sequences encoding exported proteins by using phoA gene fusions. J Bacteriol 177:59—65 Montgomery DL, Shiver JW, Leander KR et al 1993 Heterologous and homologous protection against influenza A by DNA vaccination: optimization of DNA vectors. DNA Cell Biol 12:777—783

Montgomery DL, Huygen K, Yawman AM et al 1997 Induction of humoral and cellular immune responses by vaccination with M. tuberculosis antigen 85 DNA. Cell Mol Biol 43:285—292

Murray PJ, Aldovini A, Young RA 1996 Manipulation and potentiation of antimycobacterial immunity using recombinant Bacille Calmette—Guerin strains that secrete cytokines. Proc Natl Acad Sci USA 93:934—939 Orme IM, Andersen P, Boom WH 1993 T cell response to Mycobacterium tuberculosis. J Infect Dis 67:1481—1497

Sieling, PA, Chatterjee D, Porcelli SA et al 1995 CD1-restricted T cell recognition of microbial lipoglycan antigens. Science 269:227—230 Shiver JW, Perry HC, Davies ME, Liu MA 1995 Immune responses to HIV gp120 elicited by DNA vaccination. In: Chanock RM, Brown F, Ginsberg HS, Norrby E (eds) Vaccines 95. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p 95—98 Tascon RE, Colston MJ, Ragno S, Stavropoulos E, Gregory D, Lowrie DB 1996 Vaccination against tuberculosis by DNA vaccination. Nat Med 2:888—892 Ulmer JB, Donnelly JJ, Parker SE et al 1993 Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259:1745—1749 Zhu X, Venkataprasad N, Thangaraj H et al 1997 Functions and specificity of T cells following nucleic acid vaccination of mice against Mycobacterium tuberculosis infection. J Immunol 158:5921—5926

DISCUSSION

Colston: You undoubtedly get a diverse antigen-specific immune response, and you undoubtedly see protective immune activity, but it's not clear to me that you have demonstrated that these are causally related, i.e. that the antigen-specific immune response is causing protective immunity. There are two reasons for this: (1) your protective immunity results are almost superimposable over results from my lab on six other antigens; and (2) I'm not sure that anyone, including my lab, has used a correct control. I would like to see a control experiment that looks at whether a mycobacterial protein that is not expressed in Mycobacterium tuberculosis causes protective immunity. You have shown that you get an adjuvant effect from mycobacterial DNA. Is the protection you're seeing due to that, and unrelated to the antigen-specific immune response you're observing?

Vlmer: I have to believe that at least some of the protective immunity we see is antigen specific because the control we use is the same vector without the gene insert. We have seen a DNA effect, at least in the influenza system, that can be profound on the immunogenicity of a given vector. However, in terms of protection, we've only seen slight differences between mice injected with saline and mice injected with control DNA. If the protection we saw with the Ag85

DNA was unrelated to expression of antigen we would have expected to see the same level of protection with the control DNA. We could control for any possible DNA effects in the gene itself by making a frameshift mutation, but we haven't done that. We have shown, however, that in a challenge model using a virulent human clinical isolate of M. tuberculosis a DNA vaccine encoding the mature form of Ag85, which has the same amino acid sequence as the secreted form, does not cause protective immunity.

Mi^rahi: Have you put two genes on the same plasmid to see if there are additive effects?

Jlmer: We've made dicistronic constructs, not with two different antigens, but with an antigen and a cytokine or chemokine, and showed that certain combinations can result in an enhanced immune response. But we haven't done this in the tuberculosis system, only in the influenza system.

Mi^rahi: Is it reasonable to expect that a single antigen is ever going to be as good?

Jlmer: It is likely that it won't be as good. We are already starting to see, at least in these animal models, that expression of Ag85 in isolation is not as good as Bacillus Calmette—Guerin (BCG). It might be asking too much for a single antigen to give as much protection as BCG can afford, given that BCG contains all the other antigens. We have done a lot of work on this in the influenza system. We have vaccinated mice with a combination of up to seven different DNA constructs and shown that it is possible to measure immune responses against each of these seven. They're apparently not affected by the others. Therefore, a combination vaccine for tuberculosis is feasible. We don't know whether the effects will be additive or synergistic, or whether one immunodominant protective antigen will dilute the effects of the others. Stephen Johnson has used as many as 1000—2000 discrete plasmids in combination in his expression library immunization system using Mycoplasma pulmonis as a model, and has demonstrated protection in a first round of screening (Barry et al 1995). This gives us some hope that we may be able to play with a combination of many antigens.

Kaplan: The better the immune response following improvements in the antigens used in the vaccine, the more likely the vaccine is to induce an immune response against muscle cells. This would in the long term result in muscle damage. Have you injected mice with muscle-expressing antigen for long periods of time and then looked at muscle damage? You talked about autoimmunity against DNA, but you didn't talk about autoimmunity against muscle cells.

Jlmer: We have two things in our favour: (1) we transfect few muscle cells, probably less than 1%, so we can afford to lose some; and (2) muscle cells are highly regenerative. We examined the histology of the muscle during the preclinical safety studies that were performed on the clinical flu DNA vaccines, and other people have looked at anti-muscle responses, but no-one has reported antibodies against muscle proteins, even up to two years following the injection.

Beyers: Is it possible to coexpress the co-stimulatory molecule B7 in muscle cells in order to enhance their function as antigen-presenting cells?

U/mer: People have tried to do that. We modelled our bone marrow chimera studies on the work of Drew Pardoll, who showed that in implanted tumours expressing foreign antigens antigen-presenting cells (APCs) are required and tumour cells are not converted into APCs. People have co-injected DNA with DNA expressing and B7.1 or B7.2, and have seen modest rises in antibody titres in some cases (Conry et al 1996, Iwasaki et al 1997, Tsuji et al 1997). It's unlikely that muscle cells are converted into APCs under these circumstances; but it is possible that B7 is not expressed on the same cell as the antigen is being expressed.

Brennan: You suggested that since the Ag85 has an asparagine in a glycosylation-competent motif that the expressed protein was glycosylated. Perhaps you should therefore look at an M. tubercu/osis antigen that doesn't have an asparagine in a glycosylation-competent motif. The consequences of glycosylation on the APCs could be profound because there are carbohydrate-binding proteins on the surface of these cells.

U/mer: We hypothesize that the antigens are transferred in the form of peptides, so unless the carbohydrate interferes with the processing of the protein into peptides then it probably won't be a problem. If, however, that amino acid is in the middle of the T cell epitope, it would probably interfere with the binding of the peptide either to the chaperone or MHC class I molecule. One could change the asparagine residue to glutamine in order to prevent the addition of the carbohydrate. However, although the addition would be prevented, the amino acid change itself may have a direct effect on the potency of the T cell epitope. We should consider that protein modifications that are not normally present may interfere with processing and presentation.

Hopewe//: You mentioned that you were interested in routes of administration. Have you looked at whether different routes target APCs more effectively?

U/mer: In the influenza model we looked at various routes of administration with a syringe, and we found that an immune response could be generated by intradermal and intravenous administration in addition to intramuscular administration. However, we found that protection based on cellular immune responses was superior for intramuscular injection. It has been reported that certain cationic lipids formulated with DNA can give rise to expression throughout the respiratory epithelial tract (Stribling et al 1992), so we have recently been looking at non-aerosol intranasal administration of naked DNA or lipid-modified DNA. From a vaccine point of view, it may be simplest for now to stick with intramuscular injection because that's how most vaccines are administered. However, oral and intranasal routes are also worth considering.

Rook: In view of some of the hypotheses about how peripheral tolerance to specific antigens is maintained, it may be precisely because the muscle cells are not converted into APCs that autoimmunity is not a problem. Additional tricks to turn the muscle cells into fully professional APCs should probably be avoided.

Ryfell: What sort of safety tests are carried out on DNA vaccines? Also, in your presentation you mentioned the term 'genotoxicity'. Could you explain what you mean by this?

Jlmer: DNA vaccines will undergo the same rigorous safety tests as those that have been carried out for any other vaccine on the market, as well as those that are specific for DNA vaccines, such as integration and autoimmune responses. What I mean by the term 'genotoxicity' is the degree of physical DNA changes, such as chromosomal DNA strand breaks, which are known to be induced by certain viral vaccines. This is a standard assay that is applied to the safety testing of vaccines.

Ryfell: Do you carry out any studies in non-human primates?

Jlmer: Yes. It is necessary to show in non-human primates that the construct expresses a protein and that an immune response is made against it, even if there is no challenge model.

Ryfell: In your opinion, which cytokines should be included in multiple antigenic construct vectors?

Jlmer: People have looked at many cytokines and chemokines in various models. Granulocyte macrophage colony-stimulating factor gives a modest reproducible enhancement of antibody titres, and in some cases cellular responses. This is also true for interleukin (IL)-12, IL-2 and certain chemokines, but nothing so far has been shown to give a profound response. We have found that, in general, it is difficult to improve DNA vaccines that consist of highly expressing vectors encoding protein antigens that are inherently immunogenic, whereas incremental enhancements can be made if you use a limiting dose of DNA and less than optimal vector encoding a poorly immunogenic protein.

Blackwell: In the dose—response experiments where you were testing the effect of DNA on T cell responses did you keep the total amount of DNA constant at 100 ^g? I was wondering whether you need that much DNA not because you need a certain amount of specific antigen activity but because you need a certain amount of adjuvant activity provided by non-antigen-specific sequences in the vector DNA.

Jlmer: The amount of DNA in those experiments was not constant, rather we were titrating the DNA dose, and the response was antigen specific. We found that for the influenza constructs robust responses could be observed with only 1 ^g administered once. Better responses were not always observed just by adding extra DNA. We haven't done these experiments with tuberculosis DNA vaccines, so I don't know whether this would give the same effect.

~B>lackwell: Have you tried pooling DNA vaccines for a series of unknown genes?

Vlmer: These experiments are currently in progress. There are advantages with using fewer constructs because you can keep the dose of each up in the 100 ^g range, whereas if you use 1000 constructs then the dose of each would be in the nanogram range, which may not be sufficient to generate an adequate response.

Colston: Have you treated an established infection with an antigen-specific response? Also, do you give multiple injections in the protection experiments, and if so why?

Vlmer: We haven't done many dosing experiments in the tuberculosis challenge model because of limitations on how many different things can be tested at once. In the influenza model we found that we could give a smaller total DNA dose if we administered it in multiple injections. We did not get the same type of boosting response that we do with a subunit-type vaccine, but we did observe incremental increases in antibody titres. However, the titres normalize with time, irrespective ofthe total dose or how many doses are given. The kinetics ofthe responses appear different, so it is possible that the maturation of the protective immune response is more rapid with high doses and multiple injections of DNA.

Rook: I would like to ask a question that may be unfair because we're on the record. In view of the fact that it is difficult to do vaccination trials in human tuberculosis, and in view of the fact that the efficacy of the BCG vaccine is variable, so you would be superimposing a vaccine upon this variable response, how serious are your studies on tuberculosis?

Vlmer: That's a good question. I was actually going to ask Paul Fine a similar question because I become more and more discouraged each time I hear him speak. The prospect of a 20—30-year clinical study to establish the efficacy of a vaccine will deter even the most intrepid vaccine developer. What are the prospects of setting up a large-scale, short-term trial in order to establish some measure of superiority of a vaccine over BCG? This could allow licensure of the vaccine. A long-term, post-licensure follow-up study could then be set up to investigate variations in regimen and boosters, etc.

Fine: The problem is that we need protection against adult pulmonary disease, whereas we generally talk about vaccinations in childhood. Protection in adults might be shown through a long-term follow-up study starting with children, or by aiming a trial at uninfected and/or recently infected adults. We are looking at a disease where the major burden lasts into old age, so we're also going to have to face the issue of the duration of protection, and whether we need boosters and how they can be evaluated. I agree that it is discouraging to think that it will take 20 or 30 years to complete 'all' these studies, and I too am concerned that it may put a damper on efforts to develop and evaluate vaccines. Your suggestion to explore large-scale, short-term trials to show superiority over BCG is interesting, but it still raises difficult issues. If we aim at childhood tuberculosis, e.g. meningitis, we are setting a difficult target for any new vaccine to improve upon, given that BCG appears to be consistently quite effective against such disease. For this reason I might favour setting up a comparative trial against adult tuberculosis in an area where BCG has shown to provide poor protection. If we can show that a new vaccine can provide any protection under such circumstances then we can play it by ear after that.

Vlmer: It seems to me that the most expeditious thing to do is to set the bar low in the first instance, although it would have to be high enough so that you could show superiority or equivalence to BCG.

'Donald: In primary childhood tuberculosis the two main manifestations that are clinically diagnosable and occur in high incidence areas are miliary tuberculosis and tuberculous meningitis. If your vaccine was effective these would be prevented, and by preventing these disseminated forms you might also prevent a certain amount of adult tuberculosis. It would only take a couple of years to find this out.

Vlmer: But you would have to do it as an adjunct to the BCG vaccine.

Donald: In the Western Cape we routinely vaccinate with BCG, and yet we have consistently high rates of tuberculosis meningitis and miliary tuberculosis. Therefore, there is certainly ample room for substituting a different vaccine for BCG in certain areas on the basis of our current figures.

Bateman: If there are ethical problems relating to administering the vaccine to paediatric age groups, it may be possible to compare the effect of the vaccine upon reactivation in adults, in both low and high prevalence areas. This could provide results within a reasonable time span.

Vlmer: We have considered whether there's a specific group within an industrialized country that we could look at. There are relatively high incidence areas within the US, but unfortunately those populations tend to be homeless and/or transient and are therefore not amenable to long-term follow-up studies. The only population that comes to mind is a prison population.

Bellamy: It seems to me that vaccines have two different aims. The first is to reduce the incidence of disease manifestations in populations that become infected. And the second is to reduce the fitness of the micro-organism so as to reduce its spread. Tuberculosis and malaria are particularly unusual diseases in that they have exerted significant effects on the human genome throughout the course of evolution. The human immune system has spent a long time attempting to develop basic mechanisms to reduce the fitness of those micro-organisms within human populations, but it hasn't been able to eradicate them. Therefore, it may be difficult to design a vaccine that will decrease the fitness of these micro-organisms, and perhaps the goal we ought to be aiming at is to reduce the manifestations of disease. In the case of malaria, perhaps what we ought to do is prevent cerebral malaria in children, and in the case of tuberculosis what we ought to do is reduce the incidence of miliary tuberculosis and tuberculous meningitis in children.

Jlmer: There is a perception that the gold standard for a vaccine is to induce immunity in the same way that an infection does, and that you can't improve upon immune responses generated by micro-organism infection. I don't entirely agree with this. If you can modulate the immune response or focus the immune response on specific antigens, so that when individuals encounter an organism they can rapidly respond to those antigens, then this can potentially result in better immunity than the micro-organism can itself induce, which as you mentioned has had plenty of time to evolve specific mechanisms to avoid detection and eradication by the immune system.

Bellamy: But malaria and tuberculosis may be different to other infectious diseases in that they are essentially parasites that sometimes cause diseases, and throughout history there has been substantial selective pressure on the human immune system to develop mechanisms of eradicating the micro-organism. The diseases for which we have developed effective vaccines may have not been around for as long, or they may not have been exposed to the same level of selective forces, because they occur in epidemics or because they have not been a cause of such high mortality rates as malaria and tuberculosis.

Fine: I question the idea of developing a new vaccine against tuberculous meningitis and miliary disease because most of the evidence suggests that BCG is effective against these forms of tuberculosis. In South Africa there seems to be a general feeling that BCG isn't protecting against tuberculosis meningitis, but BCG is administered in a rather peculiar way in South Africa, so many people question whether this is responsible for the failure of BCG here. Let us also not forget that tuberculous meningitis and miliary disease are rare, and these are not why tuberculosis is considered a global emergency.

Ryfell: What is different about BCG administration in South Africa?

'Donald: It's the same procedure as that used in France, Japan and in several other countries. There are arguments about the effectiveness of the precise instrument that has been used here in the past. There may be something wrong with the method of administration; however, we can certainly document whether or not children have received their BCG injection, and we find that 90% of children who present with tuberculosis meningitis have received it.

Fine: They may have received BCG but many people have questioned whether administration with this instrument is an effective way to deliver the vaccine. To my knowledge there have been no formal studies comparing its effectiveness with other methods of delivering the vaccine.

Ress: A study to address this question is due to commence shortly.

Colston: The two most recent trials where BCG has failed to protect are the Malawi and South India trials (Karonga Prevention Trial Group 1996, Baily 1980), and these are areas where BCG has been used extensively for many years. Is it possible that there has been selection for M. tuberculosis strains that are unaffected by BCG?

Fine: An interesting question. The South India BCG trial commenced in 1968 in an area where BCG hadn't previously been used, and our trial in Malawi was carried out in a population where BCG had been introduced in 1975 and had not been widely used. I would therefore be surprised if there had been selection against certain strains of M. tuberculosis, given that most transmission is thought to be attributable to adults with pulmonary disease who, in the case of these two populations, were in general born and infected before the introduction of BCG.

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