Inflammatory properties and adjuvant potential of synthetic glycolipids homologous to mycolate esters of the cell wall of Mycobacterium tuberculosis.Hermann Giresse TIMA (1), Juma'a Raheem AL DULAYYMI (2), Olivier DENIS (1), Pauline LEHEBEL (1), Klarah Sherzad BAOLS (2), Mohsin Omar MOHAMMED (2), Laurent L'HOMME (3), Mohaned Mohammed SAHB (2), Georges POTEMBERG (1), Sylvie LEGRAND (3), Roland LANG (4), Rudi BEYAERT (5), Jacques PIETTE (3), MarkStephen BAIRD (2)#, Kris HUYGEN (1)#, Marta ROMANO (1)*#.

MALT1 is involved in the activation of immune responses, as well as in the proliferation and survival of certain cancer cells. MALT1 acts as a scaffold protein for NF-κB signaling and a cysteine protease that cleaves substrates, further promoting the expression of immunoregulatory genes. Deregulated MALT1 activity has been associated with autoimmunity and cancer, implicating MALT1 as a new therapeutic target. Although MALT1 deficiency has been shown to protect against experimental autoimmune encephalomyelitis, nothing is known about the impact of MALT1 on virus infection in the central nervous system. Here, we studied infection with an attenuated rabies virus, Evelyn-Rotnycki-Abelseth (ERA) virus, and observed increased susceptibility with ERA virus in MALT1−/− mice. Indeed, after intranasal infection with ERA virus, wild-type mice developed mild transient clinical signs with recovery at 35 days postinoculation (dpi). Interestingly, MALT1−/− mice developed severe disease requiring euthanasia at around 17 dpi. A decreased induction of inflammatory gene expression and cell infiltration and activation was observed in MALT1−/− mice at 10 dpi compared to MALT1+/+ infected mice. At 17 dpi, however, the level of inflammatory cell activation was comparable to that observed in MALT1+/+ mice. Moreover, MALT1−/− mice failed to produce virus-neutralizing antibodies. Similar results were obtained with specific inactivation of MALT1 in T cells. Finally, treatment of wild-type mice with mepazine, a MALT1 protease inhibitor, also led to mortality upon ERA virus infection. These data emphasize the importance of early inflammation and activation of T cells through MALT1 for controlling the virulence of an attenuated rabies virus in the brain.IMPORTANCE Rabies virus is a neurotropic virus which can infect any mammal. Annually, 59,000 people die from rabies. Effective therapy is lacking and hampered by gaps in the understanding of virus pathogenicity. MALT1 is an intracellular protein involved in innate and adaptive immunity and is an interesting therapeutic target because MALT1-deregulated activity has been associated with autoimmunity and cancers. The role of MALT1 in viral infection is, however, largely unknown. Here, we study the impact of MALT1 on virus infection in the brain, using the attenuated ERA rabies virus in different models of MALT1-deficient mice. We reveal the importance of MALT1-mediated inflammation and T cell activation to control ERA virus, providing new insights in the biology of MALT1 and rabies virus infection.

This study develops an original co-infection model in mice using Brucella melitensis, the most frequent cause of human brucellosis, and Trypanosoma brucei, the agent of African trypanosomiasis. Although the immunosuppressive effects of T. brucei in natural hosts and mice models are well established, we observed that the injection of T. brucei in mice chronically infected with B. melitensis induces a drastic reduction in the number of B. melitensis in the spleen, the main reservoir of the infection. Similar results are obtained with Brucella abortus- and Brucella suis-infected mice and B. melitensis-infected mice co-infected with Trypanosoma cruzi, demonstrating that this phenomenon is not due to antigenic cross-reactivity. Comparison of co-infected wild-type and genetically deficient mice showed that Brucella elimination required functional IL-12p35/IFNγ signaling pathways and the presence of CD4+ T cells. However, the impact of wild type and an attenuated mutant of T. brucei on B. melitensis were similar, suggesting that a chronic intense inflammatory reaction is not required to eliminate B. melitensis. Finally, we also tested the impact of T. brucei infection on the course of Mycobacterium tuberculosis infection. Although T. brucei strongly increases the frequency of IFNγ+CD4+ T cells, it does not ameliorate the control of M. tuberculosis infection, suggesting that it is not controlled by the same effector mechanisms as Brucella. Thus, whereas T. brucei infections are commonly viewed as immunosuppressive and pathogenic, our data suggest that these parasites can specifically affect the immune control of Brucella infection, with benefits for the host.

Rabies virus is a neurovirulent RNA virus, which causes about 59000 human deaths each year. Treatment for rabies does not exist due to incomplete understanding of the pathogenesis. MALT1 mediates activation of several immune cell types and is involved in the proliferation and survival of cancer cells. MALT1 acts as a scaffold protein for NF-κB signaling and a cysteine protease that cleaves substrates, leading to the expression of immunoregulatory genes. Here, we examined the impact of genetic or pharmacological MALT1 inhibition in mice on disease development after infection with the virulent rabies virus strain CVS-11. Morbidity and mortality were significantly delayed in compared to mice, which was associated with a lower viral load, proinflammatory gene expression and infiltration and activation of immune cells in brain. Specific deletion of in T cells also delayed disease development while deletion in myeloid cells, neuronal cells or NK cells had no effect. Disease development was also delayed in mice treated with the MALT1 protease inhibitor mepazine, and in knock-in mice expressing a catalytically inactive MALT1 mutant protein, showing an important role of MALT1 proteolytic activity. The described protective effect of MALT1 inhibition against infection with a virulent rabies virus is the precise opposite of the sensitizing effect of MALT1 inhibition that we previously observed in case of infection with an attenuated rabies virus strain. Together, these data demonstrate that the role of immunoregulatory responses in rabies pathogenicity is dependent on virus virulence, and reveal the potential of MALT1 inhibition for therapeutic intervention. Rabies virus is a neurotropic RNA virus that causes encephalitis and still poses an enormous challenge to animal and public health. Efforts to establish reliable therapeutic strategies have been unsuccessful and are hampered by gaps in the understanding of virus pathogenicity. MALT1 is an intracellular protease that mediates the activation of several innate and adaptive immune cells in response to multiple receptors, and therapeutic MALT1 targeting is believed to be a valid approach for autoimmunity and MALT1-addicted cancers. Here, we study the impact of MALT1 deficiency on brain inflammation and disease development in response to infection of mice with the highly virulent CVS-11 rabies virus. We demonstrate that pharmacological or genetic MALT1 inhibition decreases neuroinflammation and extends the survival of CVS-11 infected mice, providing new insights in the biology of MALT1 and rabies virus infection.

Novel tuberculosis (TB)-vaccines preferably should (i) boost host immune responses induced by previous BCG vaccination and (ii) be directed against Mycobacterium tuberculosis (Mtb) proteins expressed throughout the Mtb infection-cycle. Human Mtb antigen-discovery screens identified antigens encoded by Mtb-genes highly expressed during in vivo murine infection (IVE-TB antigens). To translate these findings towards animal models, we determined which IVE-TB-antigens are recognised by T-cells following Mtb challenge or BCG vaccination in three different mouse strains. Eleven Mtb-antigens were recognised across TB-resistant and susceptible mice. Confirming previous human data, several Mtb-antigens induced cytokines other than IFN-γ. Pulmonary cells from susceptible C3HeB/FeJ mice produced less TNF-α, agreeing with the TB-susceptibility phenotype. In addition, responses to several antigens were induced by BCG in C3HeB/FeJ mice, offering potential for boosting. Thus, recognition of promising Mtb-antigens identified in humans validates across multiple mouse TB-infection models with widely differing TB-susceptibilities. This offers translational tools to evaluate IVE-TB-antigens as diagnostic and vaccine antigens.