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Lentiviral vectors (LVs) are highly efficient at inducing CD8 + T cell responses. However, LV-encoded antigens are processed inside the cytosol of antigen-presenting cells, which does not directly communicate with the endosomal major histocompatibility complex class II (MHC-II) presentation pathway. LVs are thus poor at inducing CD4 + T cell response. To overcome this limitation, we devised a strategy whereby LV-encoded antigens are extended at their N-terminal end with the MHC-II-associated light invariant chain (li), which contains an endosome-targeting signal sequence. When evaluated with an LV-encoded polyantigen composed of CD4 + T cell targets from Mycobacterium tuberculosis, intranasal vaccination in mice triggers pulmonary polyfunctional CD4 + and CD8 + T cell responses. Adjuvantation of these LVs extends the mucosal immunity to Th17 and Tc17 responses. A systemic prime and an intranasal boost with one of these LV induces protection against M. tuberculosis. This strategy improves the protective power of LVs against infections and cancers, where CD4 + T cell immunity plays an important role., Competing Interests: Declaration of interests P.C. is the founder and CSO of TheraVectys. J.L., F.A., P.A., M.-W.K., F.M., and A.N. are employees of TheraVectys. J.L., F.A., C.B., F.M., L.M., and P.C. are inventors of a pending patent directed to LV immunization able to induce CD4(+) T cells., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cyclic (di)nucleotides act as universal second messengers endogenously produced by several pathogens. Specifically, the roles of c-di-AMP in Mycobacterium tuberculosis immunity and virulence have been largely explored, although its contribution to the safety and efficacy of live tuberculosis vaccines is less understood. In this study, we demonstrate that the synthesis of c-di-AMP is negatively regulated by the M. tuberculosis PhoPR virulence system. Accordingly, the live attenuated tuberculosis vaccine candidate M. tuberculosis vaccine (MTBVAC), based on double phoP and fadD26 deletions, produces more than 25- and 45-fold c-di-AMP levels relative to wild-type M. tuberculosis or the current vaccine bacille Calmette-Guérin (BCG), respectively. Secretion of this second messenger was exclusively detected in MTBVAC but not in M. tuberculosis or in BCG. We also demonstrate that c-di-AMP synthesis during in vitro cultivation of M. tuberculosis is a growth-phase- and medium-dependent phenotype. To uncover the role of this metabolite in the vaccine properties of MTBVAC, we constructed and validated knockout and overproducing/oversecreting derivatives by inactivating the disA or cnpB gene, respectively. All MTBVAC derivatives elicited superior interleukin-1β (IL-1β) responses compared with BCG during an in vitro infection of human macrophages. However, both vaccines failed to elicit interferon β (IFNβ) activation in this cellular model. We found that increasing c-di-AMP levels remarkably correlated with a safer profile of tuberculosis vaccines in the immunodeficient mouse model. Finally, we demonstrate that overproduction of c-di-AMP due to cnpB inactivation resulted in lower protection of MTBVAC, while the absence of c-di-AMP in the MTBVAC disA derivative maintains the protective efficacy of this vaccine in mice., Competing Interests: C.M. and J.G.-A. are coinventors on the patent “Tuberculosis vaccine”. N.A., C.M., and J.G.-A. are coinventors on the patent “Compositions for use as a prophylactic agent to those at risk of infection of tuberculosis, or as secondary agents for treating infected tuberculosis patients”. Both patents were filed by the University of Zaragoza., (© 2022 The Authors.)

The pathogenic potential of Mycobacterium tuberculosis largely depends on ESX secretion systems exporting members of the multigenic Esx, Esp, and PE/PPE protein families. To study the secretion and regulation patterns of these proteins while circumventing immune cross-reactions due to their extensive sequence homologies, we developed an approach that relies on the recognition of their MHC class II epitopes by highly discriminative T cell receptors (TCRs) of a panel of T cell hybridomas. The latter were engineered so that each expresses a unique fluorescent reporter linked to specific antigen recognition. The resulting polychromatic and multiplexed imaging assay enabled us to measure the secretion of mycobacterial effectors inside infected host cells. We applied this novel technology to a large panel of mutants, clinical isolates, and host-cell types to explore the host-mycobacteria interplay and its impact on the intracellular bacterial secretome, which also revealed the unexpected capacity of phagocytes from lung granuloma to present mycobacterial antigens via MHC class II., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pathogens have evolved a range of mechanisms to counteract host defenses, notably to survive harsh acidic conditions in phagosomes. In the case of Mycobacterium tuberculosis, it has been shown that regulation of phagosome acidification could be achieved by interfering with the retention of the V-ATPase complexes at the vacuole. Here, we present evidence that M. tuberculosis resorts to yet another strategy to control phagosomal acidification, interfering with host suppressor of cytokine signaling (SOCS) protein functions. More precisely, we show that infection of macrophages with M. tuberculosis leads to granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion, inducing STAT5-mediated expression of cytokine-inducible SH2-containing protein (CISH), which selectively targets the V-ATPase catalytic subunit A for ubiquitination and degradation by the proteasome. Consistently, we show that inhibition of CISH expression leads to reduced replication of M. tuberculosis in macrophages. Our findings further broaden the molecular understanding of mechanisms deployed by bacteria to survive., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent insights into the mechanisms by which Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is recognized by cytosolic nucleotide sensors have opened new avenues for rational vaccine design. The only licensed anti-tuberculosis vaccine, Mycobacterium bovis BCG, provides limited protection. A feature of BCG is the partial deletion of the ESX-1 type VII secretion system, which governs phagosomal rupture and cytosolic pattern recognition, key intracellular phenotypes linked to increased immune signaling. Here, by heterologously expressing the esx-1 region of Mycobacterium marinum in BCG, we engineered a low-virulence, ESX-1-proficient, recombinant BCG (BCG::ESX-1 Mmar ) that induces the cGas/STING/TBK1/IRF-3/type I interferon axis and enhances AIM2 and NLRP3 inflammasome activity, resulting in both higher proportions of CD8 + T cell effectors against mycobacterial antigens shared with BCG and polyfunctional CD4 + Th1 cells specific to ESX-1 antigens. Importantly, independent mouse vaccination models show that BCG::ESX-1 Mmar confers superior protection relative to parental BCG against challenges with highly virulent M. tuberculosis., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The intracellular fate of Mycobacterium tuberculosis is a subject of long debate. In this issue of Cell Host & Microbe, three independent studies reveal the detection of cytosolic mycobacterial DNA by the nucleotidyltransferase cGAS, emphasizing the concept of cytosolic access by M. tuberculosis and its role in balancing immune-protection and immune-pathogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The genome of Mycobacterium tuberculosis (Mtb) encodes five type VII secretion systems, ESX-1 to ESX-5, most of which are associated with genes encoding PE/PPE proteins, named after their N-terminal Pro-Glu (PE) or Pro-Pro-Glu (PPE) motifs. Here, we describe the strong T cell immunogenicity of the ESX-5-encoded PE/PPE proteins, which share a large panel of cross-reactive CD4(+) epitopes with substantial numbers of their ESX-5-nonassociated PE/PPE homologs. The immunogenicity of these numerous PE/PPE proteins is dependent on their export by a functional EccD(5), the predicted transmembrane channel of the ESX-5 secretion apparatus. The Mtb Δppe25-pe19 mutant deleted for all ESX-5-associated pe and ppe genes, although highly attenuated in immunocompetent mice, remains able to induce immunity against the ESX-5-associated PE/PPE virulence factors, via cross-reactivity with their numerous homologs, and against the ESX-1 virulence factors ESAT-6/CFP-10. The Δppe25-pe19 strain is strongly protective against Mtb infection in mice and represents a potential antituberculosis vaccine candidate., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens., (Copyright © 2010 Elsevier Inc. All rights reserved.)