Typescript (photocopy) / vi, 184 leaves, [14] leaves of plates : ill.,(part col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.) Dept. of Zoology, University of Adelaide, 1984

Thesis (Ph.D.) Dept. of Zoology, University of Adelaide, 1984. / Typescript (photocopy).

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Globalement, un tiers de la population mondiale est infectée par Mycobacterium tuberculosis, l'agent infectieux de la tuberculose (TB). Fort heureusement, seuls 5 à 10 % des individus infectés développent un jour une TB active. Les individus non malades restent cependant infectés à vie, on parle d'infection latente. Chaque année, 8-10 millions nouveaux cas de tuberculose active sont recensés et M. tuberculosis est responsable de 1,5 à 2 millions de décès. Depuis plus d'une décennie, M. tuberculosis s'est étroitement associé à l'infection par le virus de l'immunodéficience humaine. Cette alliance néfaste représente une importante menace pour les pays en voie de développement, car ces 2 pathogènes déciment les forces vives de ces populations. Il faut malheureusement rajouter à ce triste tableau une fréquence grandissante de souches multi-résistantes, voire extensivement multi-résistantes. Face à ces souches, les avancées thérapeutiques du siècle dernier sont pratiquement réduites à néant. Considérant ces données, il est désormais crucial d'améliorer nos outils de dépistage de l'infection latente, de diagnostic de la maladie active, de prévention (vaccins) et de traitement. Pour atteindre ces objectifs, une des pistes est la caractérisation détaillée des réponses immunitaires. En comparant les réponses immunitaires des sujets infectés de manière latente à celles liées à la maladie active, nous pourrons peut-être comprendre certains mécanismes de protection. L'étude des réponses immunitaires induites par la « Heparin-Binding-Hemagglutinin » (HBHA) s'est faite dans cet objectif. La HBHA est une adhésine exprimée par le complexe M. tuberculosis. Elle est impliquée dans la dissémination extrapulmonaire du bacille et constitue donc un facteur de virulence. Par ailleurs, une vaccination de souris par seulement 3 doses de 5 µg de HBHA suffit à protéger de l'infection avec une efficacité comparable à celle du vaccin BCG. Chez l'homme, les sujets sains mais infectés développent d'importantes sécrétions d'interféron-gamma (IFN-γ) en réponse à cet antigène, alors que la majorité des patients tuberculeux ne le font pas. Cette différence est importante pour comprendre une des raisons d'échappement de M. tuberculosis au contrôle immunitaire. La HBHA est une protéine méthylée et la méthylation s’avère essentielle pour ses propriétés immunoprotectrices. Nos travaux présentés ici se sont axés sur deux éléments de la réponse immunitaire à la HBHA chez l'homme : d'une part, l'exploitation de la réponse périphérique d'IFN-γ à la HBHA comme outil de dépistage de l'infection latente et, d'autre part, l'étude des raisons de la faible sécrétion d'IFN-γ spécifique de la HBHA lors de la maladie active. L'évaluation de la sécrétion périphérique d'IFN-γ en réponse à la HBHA a permis de démontrer rétrospectivement que celle-ci permet de détecter plus de 90 % des sujets réagissant positivement à l'injection intradermique de tuberculine. De manière intéressante, l'utilisation d'un test commercial, le QuantiFERON TB Gold IT (QFT-IT) n'a permis de détecter que la moitié des sujets infectés sains. De notre point de vue, le QFT-IT ne peut être recommandé seul pour le dépistage systématique de l'infection latente par M. tuberculosis. De manière parallèle, un test de stimulation basé uniquement sur la sécrétion d’IFN-γ suite à une stimulation à l'ESAT-6, composant du QFT-IT, n'a pas permis d'augmenter la sensibilité, ni d'ajouter une plus-value au test basé sur la HBHA. A l'instar de l'intradermoréaction à la tuberculine, le dépistage de la maladie active reste décevant que ce soit par l'utilisation de la HBHA ou de l'ESAT-6. La TB active est caractérisée par une basse sécrétion périphérique d'IFN-γ en réponse à la stimulation par la HBHA. Cette faible sécrétion est cependant réversible, puisque un traitement efficace permet d'atteindre des taux d'IFN-γ significativement plus élevés. Ceci nous démontre qu'il s'agit d'une suppression associée à la phase active de l'infection. Nous avons d'abord évalué l'importance de la modulation de la sécrétion d'IFN-γ en réponse à la HBHA par 2 cytokines immunomodulatrices, l'interleukine-10 (IL-10) et le Transforming-Growth-Factor-Beta (TGF-ß). De manière intéressante, alors que ces 2 cytokines sont associées à l'infection par M. tuberculosis, la HBHA n'est inductrice ni d'IL-10, ni de TGF-ß. Les lymphocytes T régulateurs (Treg) expriment 2 marqueurs d'intérêt : le CD25, composant du récepteur à l'IL-2, et Foxp3, un gène régulateur majeur des cellules Treg. Ces cellules sont décrites comme suppressives de réponses immunitaires déclenchées par des antigènes du Soi et du non-Soi. Nous avons montré que la proportion de lymphocytes Treg périphériques est augmentée en cas de TB active. Par ailleurs, nous avons également démontré que ces cellules suppriment la sécrétion d'IFN-γ et la prolifération induite par la HBHA après stimulation des cellules mononucléées sanguines périphériques de patients tuberculeux in vitro. Cependant, la réponse anti-HBHA des patients tuberculeux, qui est démasquée par la déplétion des lymphocytes Treg, n'est pas dirigée contre des épitopes protecteurs. En effet, la méthylation n'influence pas leur sécrétion d'IFN-γ. De ce point de vue, les lymphocytes Treg sont impliqués dans la maladie tuberculeuse et influencent négativement les réponses dirigées contre un antigène protecteur. Cependant, il semble que la TB active soit également associée à une ignorance d'épitopes protecteurs. Enfin, nous avons également démontré qu'il était possible d'induire des lymphocytes Treg au départ de cellules sanguines périphériques de sujets infectés sains. En effet, la stimulation in vitro des cellules sanguines périphériques en présence de BCG et de TGF-ß est un moyen rapide pour induire l'apparition de lymphocytes Treg fonctionnels in vitro. Ceci nous interroge quant aux rôles des lymphocytes Treg dans la pathogenèse de la maladie. En effet, un excès de TGF-ß circulant est observé dans certaines conditions cliniques à haut-risque de TB post-primaire. De ce point de vue, les lymphocytes Treg pourraient être des acteurs déterminant dans la perte du contrôle à long terme de l'infection et, par là, pourraient être des cibles thérapeutiques d'intérêts lors de l'infection par M. tuberculosis. /Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). It is estimated approximately one third of the World’s population is infected with M. tuberculosis. Fortunately, only 5 to 10 % of the infected individuals will develop the disease throughout their life. However, the other healthy infected individuals remain infected for life: this is the latent TB infection (LTBI). Every year, 8 to 10 million new cases of TB are recorded globally, and about 2 to 3 million of people die from the disease. During the last several decades the co-infection of M. tuberculosis and the human immunodeficiency virus have worsened the picture. This dreadful association currently affects mostly the poorest people of the World. Unfortunately, bad news never stands alone. We now witness increasing emergence of multi-drug-resistant and even of extensively-multi-drug-resistant M. tuberculosis strains. Against these strains current therapeutics are virtually useless. The development of new tools for prevention (vaccines), diagnostics and treatment is crucial. In order to fulfill these objectives, detailed studies on the immune responses is one of the main tracks to explore. Indeed, the comparison of immune responses in LTBI subjects with those in TB patients may provide some clues to understand immune mechanisms of protection. Studies of the immune responses that are specific to Heparin-Binding-Hemagglutinin (HBHA) may be one of these clues. HBHA is an adhesin, which is expressed by the micro-organisms of the M. tuberculosis complex. It largely contributes to the extrapulmonary dissemination of the tubercle bacilli. Hence, HBHA may be qualified as an important virulence factor. Furthermore, vaccination of mice with three doses of only 5 µg HBHA each affords the same level of protection as vaccination with BCG. In humans, peripheral blood mononuclear cells (PBMC) from LTBI subjects secrete significant levels of IFN-γ in response to HBHA, whereas PBMC from TB patients do not. This discrepancy may be a cornerstone in the understanding of some of the mechanisms underlying the immune escape mediated by M. tuberculosis. HBHA is a methylated protein, and the methylation is crucial for its immuno-protective properties. This work focused on 2 major issues of the HBHA-specific immune response in humans: the use of the peripheral IFN-γ secretion in response to HBHA as a diagnostic tool for LTBI and the analysis of the underlying mechanisms to the low IFN-γ secretion during active TB. In our study, the measurement of HBHA-specific IFN-γ secretion resulted in the detection of more than 90 % of the tuberculin-skin-test (TST) positive LTBI. Strikingly, the QuantiFERON TB Gold IT (QFT-IT), a commercial test, failed to identify those LTBI subjects in more than 50 % of the cases. Therefore, we cannot recommend the use of QFT-IT alone instead of the TST for the detection of LTBI. Similarly, a test relying on the detection of IFN-γ secretion upon ESAT-6 stimulation, one of the antigens used in the QFT-IT, was not sufficiently sensitive for the LTBI detection, nor did it improve the sensitivity or the specificity of the HBHA-based test. In contrast to the diagnosis of LTBI, the tests based on HBHA- or ESAT-6-induced IFN-γ secretions displayed poor sensitivity for the diagnosis of active TB. During active TB, the HBHA-specific IFN-γ secretion in the periphery is low. However, this weak secretion is reversible upon effective treatment, as the IFN-γ response to HBHA is increased after completion of chemotherapy. This is strongly suggestive of an immune suppression during active disease. Therefore, we have first evaluated the role of two immunomodulatory cytokines, interleukin-10 (IL-10) and Transforming-Growth-Factor-Beta (TGF-ß), in the suppression of the HBHA-specific IFN-γ secretion. We found that neutralization of neither IL-10 nor TGF-ß with specific antibodies induced HBHA-specific IFN-γ secretion by PBMC of TB patients in vitro. In contrast, depletion of regulatory T cells (Treg) that express 2 major markers, CD25, a constituent of the IL-2 receptor, and Foxp3, a master regulatory gene, resulted in increased HBHA-specific IFN-γ secretion by the PBMC of TB patients. These cells are known to be involved in the suppression of immune responses to both Self and non-Self antigens. We further show that the size of the peripheral Treg cell population increases during active disease. In addition to suppressing the HBHA-specific IFN-γ secretion these cells suppress T cell proliferation in response to HBHA in vitro. However, even after depletion of the Treg cells, the uncovered HBHA-specific immune responses are not directed to the methylated epitopes during TB disease. Finally, we show that Treg cells can be induced (or expanded) from the PBMC of LTBI subjects. Stimulation of those PBMC with BCG in the presence of TGF-ß resulted in a quick appearance of functional Treg cells in vitro. This observation strongly suggests a role of Treg cells in the pathogenesis of TB, in particular in the progression of latency to reactivation. Interestingly, excessive concentration of TGF-ß, associated with various clinical conditions, is high risk factor for post-primary TB. Thus, Treg cells may result in the loss of immune control against latent M. tuberculosis infection. Therefore, Treg cells may represent potential therapeutic targets during M. tuberculosis infection.

Genetic immunization, also known as DNA or polynucleotide immunisation, is well documented to induce broad-based immunity in various animal models of infectious and non-infectious diseases. However, the low potency of DNA vaccines has to date precluded the development of commercial vaccines. The aim of this study was to systematically investigate a number of parameters to improve the potency of DNA vaccines for use in chickens, using a low pathogenic avian influenza (LPAI) virus as a proof-of-concept for their ability to produce a humoral immune response. The index virus used in the study was avian influenza virus A/coot/WA/2727/79 (H6N2), isolated from an apparently healthy Eurasian coot in 1979. Prior to any DNA experiments the virus was rigorously characterized. The virus strain was shown to be an H6 subtype by haemaglutination inhibition (HI) testing and as an N2 subtype by gene sequence analysis. The isolate was shown to be able to grow on MDCK cells in the absence of exogenous trypsin. It was further biologically characterized as LPAI with an intravenous pathogenicity index (IVPI) of 0.15 and a motif of 321PQAETRG328 at the cleavage site of the haemagglutinin (HA) protein. It was capable of infecting domestic chickens under experimental conditions with a low level of virus excretion via the cloaca and oropharynx following intravenous or oral and oculonasal inoculation. The full-length HA and nucleoprotein (NP) genes of this H6N2 virus were subsequently cloned into the eukaryotic expression vector VR1012 to generate VR-HA and VR-NP constructs. Six-week-old Hy-Line chickens were intramuscularly injected with either the VR-HA or VR-NP vaccine at different dose rates, with or without lipofectin as adjuvant. Minimal or no detectable antibody was produced, as measured by HI, ELISA and Western blotting-based assay, but high titres of H6-specific HI antibodies appeared 10 days after homologous virus challenge. In contrast to the empty vector controls, there was a significant difference in HI antibody titre between pre- and post-challenge in vaccinated birds, indicating some evidence for the priming effect of the DNA vaccines. Using the frequency of virus shedding as an indicator of protection, lower doses (50 or 100 ¦Ìg per chicken) of either adjuvanted VR-HA or VR-NP vaccine significantly reduced virus shedding in oropharyngeal and cloacal swabs compared to higher doses (300 or 500 ¦Ìg per chicken ) or empty vector control chickens. Although two vaccinations with naked VR-HA alone were not sufficient to induce an effective immune response against a homologous virus challenge, further repeat vaccinations and incorporation of adjuvant did lead to the generation of low to moderate HI antibody titres in some chickens and resulted in no or reduced virus shedding after challenge. Next, to examine the effect of expression vector, three different DNA vectors, pCI, pCI-neo and pVAX1 were used to clone the same HA gene and generate three DNA vaccine constructs. Once again, direct intramuscular injection of the three DNA constructs did not elicit measurable H6-specific HA antibody response in Hy-Line chickens but the 100 µg pCI-HA lipofectin adjuvanted vaccine group showed a significant increase in post-challenge HI titres from the naive control group, indicating that an anamnestic antibody response had been induced by the pCI-HA DNA vaccination. Compared with the controls, the three DNA constructs showed significantly reduced virus shedding in cloacal swabs post virus challenge, suggesting that the three DNA vaccines induced some level of immune response in vaccinated chickens. As with the VR-HA construct, the lower dose groups for each vaccine (50 or 100 g) were more effective at reducing virus shedding from the cloaca than the higher dose group (300 g). To further investigate why the DNA vaccines did not elicit a measurable antibody response, the HA gene incorporating a Kozak enhancer sequence was cloned into an alternative expression vector, pCAGGS, to produce the pCAG-HAk construct. Three-week-old SPF chickens were immunized with this construct either by the intramuscular route (IM) or electroporation (EP). H6 HI antibodies were present in some chickens by 3 weeks after the first IM vaccination and 75% of the chickens vaccinated with 10, 100 or 300 µg pCAG-HAk were antibody positive by 2 weeks after the second IM vaccination. For EP immunization, 87.5% of vaccinated birds seroconverted after the first vaccination and 100% seroconverted after the second vaccination and the H6 HI antibody titres were significantly higher than for chickens vaccinated by IM inoculation. Another group was given a single dose IM vaccination with 100 µg of the pCAG-HAk construct and showed a maximum sero-conversion rate of 53.3% with a peak H6 HI titre of 27 at 5 weeks post-vaccination. This demonstrated that optimization of the expression vector and insertion of a Kozak sequence could synergistically enhance expression of the H6 HA gene and result in a measurable H6 antibody response in SPF chickens. EP was also compared with IM inoculation with the 100 g pCI-HA construct in SPF chickens, resulting in a 50% sero-conversion rate and mean HI titre of 21.3 at 2 weeks after the second vaccination by EP. By comparison, only 25% chickens had trace HI titres by IM inoculation. This indicated that EP was more efficient than IM delivery for both constructs. A codon-optimized complete HA gene from A/coot/WA/2727/79 (H6N2) was then chemically synthesized and cloned into a pCAGGS vector to generate the pCAG-optiHAk construct. SPF chickens immunized twice with either 10 µg or 100 µg of pCAG-optHA showed 37.5% and 87.5% sero-conversion rates respectively, with a mean H6 HI tire of 21.4 and 22.6 at 3 weeks after the second immunization, but the differences were not statistically significant. There were also no significant differences in either the sero-conversion rate or the H6 HI titre between the pCAG-HAk and pCAG-optiHAk groups, suggesting that a codon-optimized HA DNA vaccine did not achieve significantly better immunogenicity than the pCAG-HAk vaccine. In vitro expression of the developed DNA constructs in chicken-, hamster-, monkey- and human-origin cells, as measured by Western blotting and immunofluorescence testing (IFT), showed the strength of H6 HA expression in the following descending order - pCAG-optiHAk/pCAG-HAk, pCI-HAk, VR-HA, pCI-HA, pCIneo-HA and pVAX-HA. The in vivo chicken vaccinations also showed that the pCI-HA construct was more effective than the pCI-neo-HA, and that the pCAG-optiHA or pCAG-HAk constructs were better than pCI-HAk in term of reduction in virus shedding after H6N2 virus challenge. Thus, in vitro HA gene expression directly correlated with the generation of immune responses in vivo, indicating that in vitro studies can be used for pre-selection of expression plasmids prior to development of avian influenza DNA vaccines. Lipofectin as a chemical adjuvant was shown to enhance the DNA-induced immune response but is prohibitively expensive for routine use in poultry vaccines. Thus, an experimental adjuvant for poultry DNA vaccines (Essai) and a new nanoparticle (Phema) adjuvant used for the first time in poultry were compared with conventional aluminum salts (alum) adjuvant in the present study. No HI antibody was detected in any adjuvant-vaccinated Hy-Line chickens following two immunizations. However, in comparison with the naive control group, the alum- and Phema adjuvanted pCAG-HAk groups significantly reduced the frequency of virus shedding in oropharyngeal swabs, but Essai adjuvant was not effective in augmenting the pCAG-HAk vaccine efficacy. This pilot study also emphasised that the traditional aluminum hydroxide adjuvant, either DNA binding or non-binding, may be useful as an adjuvant for enhancing DNA-induced immune responses in chickens owing to its low price and safety record. Overall, DNA immunization with various HA-expressing constructs was shown to be variably effective in inducing immune responses in chickens. The efficacy of DNA vaccines could be synergistically improved by taking appropriate approaches. With continuing research DNA vaccines have the potential to become an important tool for disease prevention and control.

Influenza A viruses are important pathogens of humans, other mammals and birds. Swine are considered to be the ‘mixing vessel’ for influenza viruses because of their susceptibility to infection with not only swine influenza viruses but also human and avian influenza viruses. After infection of pigs with different influenza viruses, reassortment events between genomic RNA segments and point mutations can take place which can result in novel influenza virus strains capable of causing human pandemics. To combat infections, vaccination is available in many countries for humans, but not typically used in pigs. However, anti-influenza drugs have been used to treat livestock, and mutations conferring drug resistance occur in circulating strains. The mechanisms responsible for the emergence and spread of drug resistant mutations against amantadine and oseltamivir have been studied previously but often gave conflicting results. Therefore, this PhD thesis focused on resolving the mechanisms responsible for this rapid drug resistance spread. In chapter one I examine the extent of reassortment events in swine influenza A viruses by analysing within subtype reassortment and extrapolating the results for the between subtype reassortment. Reassortment is one of the mechanisms that can be responsible for mutations, conferring resistance to drugs, to spread between strains, and thus spread in the host population. The findings of this chapter show that the genomic segments most prone to reassortment code for a polymerase (PB1) and both glycoproteins, within all three subtypes studied. Since particular mutations in the matrix protein (MP) segment cause resistance to amantadine, my study focused on MP compared to other segments and revealed moderate level of reassortment. MP reassorts well with polymerases, both within and between subtype, while nonstructural (NS) is least likely to reassort. Chapter two of this thesis aimed at resolving the origin and spread of the most common drug resistance conferring mutation in swine influenza viruses which causes amantadine resistance. I show first that this mutation occurred in swine influenza viruses and was therefore not transmitted from the recently ancestral avian influenza strains, and second that the prevalence of resistance in swine influenza viruses is due to functional linkage of mutations at other sites and not by direct drug pressure. In chapter three I examine the mechanisms responsible for the rapid rise and spread of oseltamivir resistance in human influenza H1N1 viruses which arose in the absence of drug use. The primary mutation lies in the neuraminidase glycoprotein but because of the close functional interaction I focus on changes in haemagglutinin that occurred in association with resistance. The results showed several mutations in haemagglutinin were associated with resistance suggesting selection acting on haemagglutinin in order to balance the activity of both glycoproteins. Overall these results show the importance of functional linkage between segments as a mechanism for the occurrence of drug resistance conferring mutations, and reassortment as a means of spreading these mutations into newly emerging strains.

Background P. gingivalis, a non-motile, rod-shaped, anaerobic, Gram-negative bacterium is one of the principal sources of periodontal disease. It possesses a number of potential virulence factors thought to be important in the disease process including 5 hemagglutinins (Hag). One of these is HagB. It is a well characterized nonfimbrial adhesin expressed on the surface of P. gingivalis. HagB is very pro-inflammatory and induces robust chemokine and cytokine responses in vitro and in vivo. Since the chemokine and cytokine responses seen from single cells grown in tissue culture often are not representative of the chemokine and cytokine profiles seen in clinical samples or biopsy specimens, we devised a co-culture model of keratinocytes, dendritic cells, and T-cells to test the hypothesis that chemokine and cytokine responses from co-cultured cells would be more representative of responses seen in clinical samples from individuals with periodontal disease than single cell models. Methods and materials HagB was prepared by cloning hagb of P. gingivalis (1.4 kb) into the vector pQE31 (QIAGEN Inc., Valencia, CA); expressed in E. coli M15(pREP4)pQE31-TX1; and isolated from E. coli lysates by affinity chromatography using a Ni-charged resin (Profinity IMAC Resin, BioRad, Hercules, CA) and examined by SDS-PAGE. Co-culture models were treated with 10 µg/ml HagB (Test) or 10 µg/ml HagB diluent (Control). At 64 hours the supernatants were collected. Chemokine and cytokine biomarkers GM-CSF, CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), IL-1α, IL-6, IL-8, TNFα, IL-12(p40), and VEGF responses were determined using Milliplex immunoassays. HagB responses were corrected by subtracting the constitutive responses detected in supernatants incubated with HagB diluent. Statistical differences among groups were determined on Log10 transformed biomarker concentration using JMP 10 (version 10.0, SAS, CAR; NC). Results Buffers (e.g. HagB diluent) did not induce a chemokine or cytokine response, however there was a gradual increase in chemokine and cytokine responses from cells at 64 hours. These were subtracted from HagB induced responses. Responses generally fell in 2 groups; in one group containing VEGF, IL-12(p40), IL-6, RANTES and GM-CSF, there were no significant differences among groups (p>0.05). In another group containing IL-1α, IL-8, MIP-1α, MIP-1β and TNF-α, there were significant differences among groups (p< 0.05). Interestingly these resulting responses fell in 2 categories- GM-CSF, IL-12, IL-1α, IL-6 were less than 25pg/ml and IL-8, MIP-1α, MIP-1β, RANTES, TNFα and VEGF were more than 25pg/ml. Some responses were driven by a particular cell type e.g. GM-CSF produced by dendritic cells, RANTES produced by T- cells and VEGF produced by T cells. There were similar responses in HagB-induced IL-8, MIP-1β, MIP-1α and TNFα responses by dendritic cells + keratinocytes and dendritic cells + keratinocytes + T cells. Conclusions Co-culture models can more realistically produce chemokine and cytokine responses to agonists than individual cultures of cells, which is important for predicting and assessing novel therapeutic treatments of periodontal disease.

Globalement, un tiers de la population mondiale est infectée par Mycobacterium tuberculosis, l'agent infectieux de la tuberculose (TB). Fort heureusement, seuls 5 à 10 % des individus infectés développent un jour une TB active. Les individus non malades restent cependant infectés à vie, on parle d'infection latente. Chaque année, 8-10 millions nouveaux cas de tuberculose active sont recensés et M. tuberculosis est responsable de 1,5 à 2 millions de décès. Depuis plus d'une décennie, M. tuberculosis s'est étroitement associé à l'infection par le virus de l'immunodéficience humaine. Cette alliance néfaste représente une importante menace pour les pays en voie de développement, car ces 2 pathogènes déciment les forces vives de ces populations. Il faut malheureusement rajouter à ce triste tableau une fréquence grandissante de souches multi-résistantes, voire extensivement multi-résistantes. Face à ces souches, les avancées thérapeutiques du siècle dernier sont pratiquement réduites à néant. <p>Considérant ces données, il est désormais crucial d'améliorer nos outils de dépistage de l'infection latente, de diagnostic de la maladie active, de prévention (vaccins) et de traitement. Pour atteindre ces objectifs, une des pistes est la caractérisation détaillée des réponses immunitaires. En comparant les réponses immunitaires des sujets infectés de manière latente à celles liées à la maladie active, nous pourrons peut-être comprendre certains mécanismes de protection. L'étude des réponses immunitaires induites par la « Heparin-Binding-Hemagglutinin » (HBHA) s'est faite dans cet objectif. La HBHA est une adhésine exprimée par le complexe M. tuberculosis. Elle est impliquée dans la dissémination extrapulmonaire du bacille et constitue donc un facteur de virulence. Par ailleurs, une vaccination de souris par seulement 3 doses de 5 µg de HBHA suffit à protéger de l'infection avec une efficacité comparable à celle du vaccin BCG. Chez l'homme, les sujets sains mais infectés développent d'importantes sécrétions d'interféron-gamma (IFN-γ) en réponse à cet antigène, alors que la majorité des patients tuberculeux ne le font pas. Cette différence est importante pour comprendre une des raisons d'échappement de M. tuberculosis au contrôle immunitaire. La HBHA est une protéine méthylée et la méthylation s’avère essentielle pour ses propriétés immunoprotectrices. <p>Nos travaux présentés ici se sont axés sur deux éléments de la réponse immunitaire à la HBHA chez l'homme :d'une part, l'exploitation de la réponse périphérique d'IFN-γ à la HBHA comme outil de dépistage de l'infection latente et, d'autre part, l'étude des raisons de la faible sécrétion d'IFN-γ spécifique de la HBHA lors de la maladie active.<p> <p>L'évaluation de la sécrétion périphérique d'IFN-γ en réponse à la HBHA a permis de démontrer rétrospectivement que celle-ci permet de détecter plus de 90 % des sujets réagissant positivement à l'injection intradermique de tuberculine. De manière intéressante, l'utilisation d'un test commercial, le QuantiFERON TB Gold IT (QFT-IT) n'a permis de détecter que la moitié des sujets infectés sains. De notre point de vue, le QFT-IT ne peut être recommandé seul pour le dépistage systématique de l'infection latente par M. tuberculosis. De manière parallèle, un test de stimulation basé uniquement sur la sécrétion d’IFN-γ suite à une stimulation à l'ESAT-6, composant du QFT-IT, n'a pas permis d'augmenter la sensibilité, ni d'ajouter une plus-value au test basé sur la HBHA. A l'instar de l'intradermoréaction à la tuberculine, le dépistage de la maladie active reste décevant que ce soit par l'utilisation de la HBHA ou de l'ESAT-6.<p>La TB active est caractérisée par une basse sécrétion périphérique d'IFN-γ en réponse à la stimulation par la HBHA. Cette faible sécrétion est cependant réversible, puisque un traitement efficace permet d'atteindre des taux d'IFN-γ significativement plus élevés. Ceci nous démontre qu'il s'agit d'une suppression associée à la phase active de l'infection. Nous avons d'abord évalué l'importance de la modulation de la sécrétion d'IFN-γ en réponse à la HBHA par 2 cytokines immunomodulatrices, l'interleukine-10 (IL-10) et le Transforming-Growth-Factor-Beta (TGF-ß). De manière intéressante, alors que ces 2 cytokines sont associées à l'infection par M. tuberculosis, la HBHA n'est inductrice ni d'IL-10, ni de TGF-ß. Les lymphocytes T régulateurs (Treg) expriment 2 marqueurs d'intérêt :le CD25, composant du récepteur à l'IL-2, et Foxp3, un gène régulateur majeur des cellules Treg. Ces cellules sont décrites comme suppressives de réponses immunitaires déclenchées par des antigènes du Soi et du non-Soi. Nous avons montré que la proportion de lymphocytes Treg périphériques est augmentée en cas de TB active. Par ailleurs, nous avons également démontré que ces cellules suppriment la sécrétion d'IFN-γ et la prolifération induite par la HBHA après stimulation des cellules mononucléées sanguines périphériques de patients tuberculeux in vitro. Cependant, la réponse anti-HBHA des patients tuberculeux, qui est démasquée par la déplétion des lymphocytes Treg, n'est pas dirigée contre des épitopes protecteurs. En effet, la méthylation n'influence pas leur sécrétion d'IFN-γ. De ce point de vue, les lymphocytes Treg sont impliqués dans la maladie tuberculeuse et influencent négativement les réponses dirigées contre un antigène protecteur. Cependant, il semble que la TB active soit également associée à une ignorance d'épitopes protecteurs.<p>Enfin, nous avons également démontré qu'il était possible d'induire des lymphocytes Treg au départ de cellules sanguines périphériques de sujets infectés sains. En effet, la stimulation in vitro des cellules sanguines périphériques en présence de BCG et de TGF-ß est un moyen rapide pour induire l'apparition de lymphocytes Treg fonctionnels in vitro. Ceci nous interroge quant aux rôles des lymphocytes Treg dans la pathogenèse de la maladie. En effet, un excès de TGF-ß circulant est observé dans certaines conditions cliniques à haut-risque de TB post-primaire. De ce point de vue, les lymphocytes Treg pourraient être des acteurs déterminant dans la perte du contrôle à long terme de l'infection et, par là, pourraient être des cibles thérapeutiques d'intérêts lors de l'infection par M. tuberculosis. /Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). It is estimated approximately one third of the World’s population is infected with M. tuberculosis. Fortunately, only 5 to 10 % of the infected individuals will develop the disease throughout their life. However, the other healthy infected individuals remain infected for life: this is the latent TB infection (LTBI). Every year, 8 to 10 million new cases of TB are recorded globally, and about 2 to 3 million of people die from the disease. During the last several decades the co-infection of M. tuberculosis and the human immunodeficiency virus have worsened the picture. This dreadful association currently affects mostly the poorest people of the World. Unfortunately, bad news never stands alone. We now witness increasing emergence of multi-drug-resistant and even of extensively-multi-drug-resistant M. tuberculosis strains. Against these strains current therapeutics are virtually useless. <p>The development of new tools for prevention (vaccines), diagnostics and treatment is crucial. In order to fulfill these objectives, detailed studies on the immune responses is one of the main tracks to explore. Indeed, the comparison of immune responses in LTBI subjects with those in TB patients may provide some clues to understand immune mechanisms of protection. Studies of the immune responses that are specific to Heparin-Binding-Hemagglutinin (HBHA) may be one of these clues. HBHA is an adhesin, which is expressed by the micro-organisms of the M. tuberculosis complex. It largely contributes to the extrapulmonary dissemination of the tubercle bacilli. Hence, HBHA may be qualified as an important virulence factor. Furthermore, vaccination of mice with three doses of only 5 µg HBHA each affords the same level of protection as vaccination with BCG. In humans, peripheral blood mononuclear cells (PBMC) from LTBI subjects secrete significant levels of IFN-γ in response to HBHA, whereas PBMC from TB patients do not. This discrepancy may be a cornerstone in the understanding of some of the mechanisms underlying the immune escape mediated by M. tuberculosis. HBHA is a methylated protein, and the methylation is crucial for its immuno-protective properties. <p>This work focused on 2 major issues of the HBHA-specific immune response in humans: the use of the peripheral IFN-γ secretion in response to HBHA as a diagnostic tool for LTBI and the analysis of the underlying mechanisms to the low IFN-γ secretion during active TB.<p> <p>In our study, the measurement of HBHA-specific IFN-γ secretion resulted in the detection of more than 90 % of the tuberculin-skin-test (TST) positive LTBI. Strikingly, the QuantiFERON TB Gold IT (QFT-IT), a commercial test, failed to identify those LTBI subjects in more than 50 % of the cases. Therefore, we cannot recommend the use of QFT-IT alone instead of the TST for the detection of LTBI. Similarly, a test relying on the detection of IFN-γ secretion upon ESAT-6 stimulation, one of the antigens used in the QFT-IT, was not sufficiently sensitive for the LTBI detection, nor did it improve the sensitivity or the specificity of the HBHA-based test. In contrast to the diagnosis of LTBI, the tests based on HBHA- or ESAT-6-induced IFN-γ secretions displayed poor sensitivity for the diagnosis of active TB.<p>During active TB, the HBHA-specific IFN-γ secretion in the periphery is low. However, this weak secretion is reversible upon effective treatment, as the IFN-γ response to HBHA is increased after completion of chemotherapy. This is strongly suggestive of an immune suppression during active disease. Therefore, we have first evaluated the role of two immunomodulatory cytokines, interleukin-10 (IL-10) and Transforming-Growth-Factor-Beta (TGF-ß), in the suppression of the HBHA-specific IFN-γ secretion. We found that neutralization of neither IL-10 nor TGF-ß with specific antibodies induced HBHA-specific IFN-γ secretion by PBMC of TB patients in vitro. In contrast, depletion of regulatory T cells (Treg) that express 2 major markers, CD25, a constituent of the IL-2 receptor, and Foxp3, a master regulatory gene, resulted in increased HBHA-specific IFN-γ secretion by the PBMC of TB patients. These cells are known to be involved in the suppression of immune responses to both Self and non-Self antigens. We further show that the size of the peripheral Treg cell population increases during active disease. In addition to suppressing the HBHA-specific IFN-γ secretion these cells suppress T cell proliferation in response to HBHA in vitro. However, even after depletion of the Treg cells, the uncovered HBHA-specific immune responses are not directed to the methylated epitopes during TB disease. <p>Finally, we show that Treg cells can be induced (or expanded) from the PBMC of LTBI subjects. Stimulation of those PBMC with BCG in the presence of TGF-ß resulted in a quick appearance of functional Treg cells in vitro. This observation strongly suggests a role of Treg cells in the pathogenesis of TB, in particular in the progression of latency to reactivation. Interestingly, excessive concentration of TGF-ß, associated with various clinical conditions, is high risk factor for post-primary TB. Thus, Treg cells may result in the loss of immune control against latent M. tuberculosis infection. Therefore, Treg cells may represent potential therapeutic targets during M. tuberculosis infection. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished