Role of Interleukin-17A in the immune response to Mycobacterium tuberculosis in mice and vaccine applications

La tuberculose est une maladie contagieuse causée par une infection avec M. tuberculosis. Son incidence globale élevée, des traitements longs et coûteux, l’apparition de souches résistantes aux antibiotiques disponibles et la co-infection avec le VIH en font un problème de santé publique de premier plan. En effet, l’Organisation Mondiale de la Santé estime qu’un tiers de la population mondiale est infectée de façon latente par M. tuberculosis. La mise au point d’un vaccin efficace serait un des moyens de mieux contrôler cette maladie et pour cela une meilleure compréhension de la réponse immunitaire contre M. tuberculosis est indispensable. Il est cependant clair que la réponse Th1 et l’IFN-γ sont essentiels pour la protection contre M. tuberculosis. Cependant, beaucoup d’aspects de cette immunité restent encore indéterminés dont le rôle de la réponse IL-17A. Dans ce travail, nous avons analysé la susceptibilité de souris génétiquement déficientes pour la sous-unité A du récepteur de l’IL-17A à une infection par M. tuberculosis. Nous montrons que la signalisation induite par l’IL-17A est indispensable pour le contrôle à long terme de l’infection et ce malgré une augmentation de la réponse IFN-γ.Dans la deuxième partie du travail, nous avons analysé l’effet de la neutralisation de l’IL-12 sur la susceptibilité de souris préalablement vaccinées avec le Bacille de Calmette et Guérin (BCG) à une infection par M. tuberculosis. La neutralisation de l’IL-12 a été réalisée en utilisant un auto-vaccin anti-IL-12. Les résultats ont confirmé le rôle essentiel de l’IL-12 dans la protection contre une infection primaire avec M. tuberculosis ;ils ont cependant également permis de démontrer que la neutralisation de l’IL-12 n’exerce qu’un effet très modeste sur la protection conférée par le vaccin BCG. Ainsi, la diminution d’IFN-γ induite par la neutralisation de l’IL-12 semble être compensée par une augmentation de la production de TNF-α, d’IL-6 et plus particulièrement de l’IL-17A.En conclusion, notre travail indique que la réponse IL-17A est importante pour la protection contre M. tuberculosis que ce soit lors d’une infection primaire ou en cas de réponse mémoire. De plus, nos observations renforcent l’idée de plus en plus communément admise que l’IFN-γ seul n’est pas suffisant pour protéger contre M. tuberculosis/Tuberculosis is a contagious disease caused by infection with M. tuberculosis. Due to its high global incidence, the length and cost of antibiotic treatments, the emergence of antibiotics resistant strains and co-infection with HIV, Tuberculosis remains a major health problem. In addition, World Health Organization estimates that one-third of the world population is latently infected with M. tuberculosis. The development of an efficient vaccine could lead to a better control of this disease, but for that purpose a better understanding of the protective immune response against M. tuberculosis is essential. It is clear that Th1 immunity and IFN-γ play an essential role in protection against M. tuberculosis. However, numerous aspects of this immune response are still poorly understood, such as the role of the IL-17A response. In this work, we have analyzed the susceptibility to M. tuberculosis infection in mice genetically inactivated in the IL-17 receptor.A subunit. We have shown that IL-17A signalling is required for long-term control of M. tuberculosis infection, even if the IFN-γ response is increased. In the second part of this work, we have analyzed the effect of IL-12 in resistance against M. tuberculosis infection and in the protection conferred by the BCG vaccine. For that purpose, IL-12 was neutralized using an anti-IL-12 auto-vaccine. Our results confirm the essential role of IL-12 in the protection against a primary M. tuberculosis infection. Nevertheless, these results also demonstrate that IL-12 neutralization only marginally affect the protection conferred by the BCG vaccine. Indeed, the decreased IFN-γ production induced by IL-12 neutralization in BCG-vaccinated mice seems compensated by increased TNF-α, IL-6 and more specifically IL-17A production.In conclusion, our data indicate that the IL-17A response is important in protection against M. tuberculosis, both in primary infection or in the case of memory responses. Moreover, our results emphasize the emerging idea that a functional IFN-γ response alone is not sufficient to protect against M. tuberculosis.Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

PEGylation prolongs the pulmonary retention of an anti-IL-17A Fab' antibody fragment after pulmonary delivery in three different species.

The PEGylation of antibody fragments has been shown to greatly prolong their residence time in the lungs in mice. The purpose of this research was to confirm the effect of PEGylation in higher animal species, that is, the rat and the rabbit. An anti-IL-17A Fab' antibody fragment was conjugated to a two-armed 40kDa polyethylene glycol (PEG) via site-selective thiol PEGylation. PEGylation did not significantly alter the binding activity of the Fab' fragment but it largely enhanced its inhibitory potency. PEGylation increased the residence time of the Fab' in the lungs of mice, rats and rabbits. Following intratracheal administration, the unconjugated Fab' was cleared from the lungs within 24h while large quantities of the PEGylated Fab' remained present up to 48h. No significant differences in clearance were noted between the three animal species although there was a tendency of longer residence time in higher species. PEGylation represents a promising approach to sustain the presence of antibody fragments in the lungs and to enhance their therapeutic efficacy in respiratory diseases

Preclinical evaluation of topically-administered PEGylated Fab'lung toxicity

PEGylation is a promising approach to increase the residence time of antibody fragments in the lungs and sustain their therapeutic effects. However, concerns arise as to the potential pulmonary toxicity of antibody fragments conjugated to high molecular weight (HMW)polyethylene glycol (PEG), notably after repeated administrations, and the possibility of PEG accumulation in the lungs. The purpose of this proof-of-concept study is to give insights about the safety of lung administration of a Fab’ anti-IL17A antibody fragment conjugated to two-armed 40kDa PEG (PEG40). The presence of the PEG40 moiety inside alveolar macrophages remained stable for at least 24 hours after intratracheal administration of PEG40-Fab’ to mice. PEG40 was then progressively cleared from alveolar macrophages. Incubation of PEG40 alone with macrophages in vitro did not significantly harm macrophages and did not affect phagocytosis or the production of inflammatory markers. After acute or chronic administration of PEG40-Fab’ to mice, no signs of significant pulmonary toxicity or inflammatory cell accumulation were observed. A vacuolization of alveolar macrophages not associated with any inflammation was noticed when PEG40, PEG40-Fab’, or unPEGylated Fab’ were administered. To conclude this preliminary proof of concept study, acute or repeated pulmonary administrations of PEGylated Fab’ appear safe in rodents

Increased pulmonary tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-17A responses compensate for decreased gamma interferon production in anti-IL-12 autovaccine-treated, Mycobacterium bovis BCG-vaccinated mice.

Interleukin-12 (IL-12) and IL-23 (which share a p40 subunit) are pivotal cytokines in the generation of protective Th1/Th17-type immune responses upon infection with the intracellular pathogen Mycobacterium tuberculosis. The role of IL-12 and IL-23 in protection conferred by the tuberculosis vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) is, however, less well documented. By using an autovaccine approach, i.e., IL-12p70 cross-linked with ovalbumin and PADRE peptide formulated with the GSK proprietary adjuvant system AS02(V), we could specifically neutralize IL-12 while leaving the IL-23 axis intact. Neutralization of IL-12 before M. tuberculosis challenge rendered C57BL/6 mice highly susceptible, resulting in 30-fold-higher CFU in spleen and lungs and accelerated mortality. In contrast, neutralization of IL-12 in BCG-vaccinated mice prior to M. tuberculosis challenge only marginally affected vaccine-mediated protection. Analysis of cytokine production in spleen and lungs 3 weeks post-TB challenge by enzyme-linked immunosorbent assay and functional and flow cytometric assays showed significantly reduced mycobacterium-specific gamma interferon (IFN-γ) responses in M. tuberculosis-infected and BCG-vaccinated mice that had been treated with the autovaccine. Purified protein derivative-induced tumor necrosis factor alpha (TNF-α), IL-6, and IL-17A levels, however, were highest in lungs from BCG-vaccinated/IL-12-neutralized animals, and even unstimulated lung cells from these mice produced significant levels of the three cytokines. Mycobacterium-specific IL-4 and IL-5 production levels were overall very low, but IL-12 neutralization resulted in increased concanavalin A-triggered polyclonal secretion of these Th2-type cytokines. These results suggest that TNF-α, IL-6, and IL-17A may be more important pulmonary effector molecules of BCG-mediated protection than IFN-γ in a context of IL-12 deficiency

Fate of PEGylated antibody fragments following delivery to the lungs: Influence of delivery site, PEG size and lung inflammation.

Pulmonary administration of anti-cytokine antibodies offers a targeted therapy in asthma. However, the rapid elimination of proteins from the lungs limits the efficacy of inhaled medications. PEGylation has been shown to increase the residence time of anti-interleukin (IL)-17A and anti-IL-13 antibody fragments in the lungs and to improve their therapeutic efficacy. Yet, little is known about the factors that affect the residence time of PEGylated antibody fragments in the lungs following pulmonary delivery. In this study, we showed that the molecular weight of polyethylene glycol (PEG), 20kDa or 40kDa, had a moderate effect on the residence time of an anti-IL-17A Fab' fragment in the lungs of mice. By contrast, the site of delivery of the anti-IL-17A and anti-IL-13 Fab' fragments within the lungs had a major impact on their residence time, with the deeper the delivery, the more prolonged the residence time. The nature of the Fab' fragment had an influence on its residence time as well and the anti-IL-17A Fab' benefited more from PEGylation than the anti-IL-13 Fab' did. Acute lung inflammation slightly shortened the residence time of the anti-IL-17A and anti-IL-13 Fab' fragments in the lungs but PEGylation was able to prolong their presence in both the healthy and inflamed lungs. Antibody fragments were predominately located within the airway lumen rather than the lung parenchyma. Transport experiments on monolayers of Calu-3 cells and studies of fluorescence recovery after photobleaching in respiratory mucus showed that mechanisms involved in the prolonged presence of PEGylated Fab' in the airway lumen might include binding to the mucus, reduced uptake by respiratory cells and reduced transport across lung epithelia. Finally, using I125-labeled anti-IL-17A Fab', we showed that the protein fragment hardly penetrated into the lungs following subcutaneous injection, as opposed to pulmonary delivery