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Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisΔRD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective.

Mycobacterium tuberculosis (Mtb) uses efficient strategies to evade the eradication by professional phagocytes, involving--as recently confirmed--escape from phagosomal confinement. While Mtb determinants, such as the ESX-1 type VII secretion system, that contribute to this phenomenon are known, the host cell factors governing this important biological process are yet unexplored. Using a newly developed flow-cytometric approach for Mtb, we show that macrophages expressing the phagosomal bivalent cation transporter Nramp-1, are much less susceptible to phagosomal rupture. Together with results from the use of the phagosome acidification inhibitor bafilomycin, we demonstrate that restriction of phagosomal acidification is a prerequisite for mycobacterial phagosomal rupture and cytosolic contact. Using different in vivo approaches including an enrichment and screen for tracking rare infected phagocytes carrying the CD45.1 hematopoietic allelic marker, we here provide first and unique evidence of M. tuberculosis-mediated phagosomal rupture in mouse spleen and lungs and in numerous phagocyte types. Our results, linking the ability of restriction of phagosome acidification to cytosolic access, provide an important conceptual advance for our knowledge on host processes targeted by Mtb evasion strategies.

Mutated oncogenic Kirsten rat sarcoma virus (KRAS) antigen is expressed in a large variety of cancers, including pancreatic, colorectal, and pulmonary cancers. The oncogenic KRAS mutations cause malignancies and are usually ubiquitously expressed by all cells of a tumor. The KRAS amino acid substitutions at the positions 12 or 13 are among the most frequent mutations in human cancers. Here, we developed immuno-oncotherapeutic non-integrative lentiviral vectors encoding a segment encompassing KRAS G12D , either alone, or associated with antigen carriers. These carriers can improve the intracellular antigen routing to major histocompatibility complex presentation machineries or provide universal helper CD4 + epitopes. Immunotherapy with one of these vectors resulted in significant immune control of tumor growth in colorectal or pulmonary preclinical cancer models, in several murine genetic backgrounds. The antitumor effect was correlated with increased proportions of intra-tumoral hematopoietic cells and notably CD8 + T cells. Although this effect was partial, it was robust, reproducible and advantageously combinable with conventional chemotherapies and immunotherapies to improve antitumor protection. Therefore, this approach shows promise as an immuno-oncotherapy against KRAS-mediated malignant transformation.

Tuberculosis (TB) is a leading infectious cause of death worldwide. The use of ethionamide (ETH), a main second line anti-TB drug, is hampered by its severe side effects. Recently discovered “booster” molecules strongly increase the ETH efficacy, opening new perspectives to improve the current clinical outcome of drug-resistant TB. To investigate the simultaneous delivery of ETH and its booster BDM41906 in the lungs, we co-encapsulated these compounds in biodegradable polymeric nanoparticles (NPs), overcoming the bottlenecks inherent to the strong tendency of ETH to crystallize and the limited water solubility of this Booster. The efficacy of the designed formulations was evaluated in TB infected macrophages using an automated confocal high-content screening platform, showing that the drugs maintained their activity after incorporation in NPs. Among tested formulations, “green” β-cyclodextrin (pCD) based NPs displayed the best physico-chemical characteristics and were selected for in vivo studies. The NPs suspension, administered directly into mouse lungs using a Microsprayer®, was proved to be well-tolerated and led to a 3-log decrease of the pulmonary mycobacterial load after 6 administrations as compared to untreated mice. This study paves the way for a future use of pCD NPs for the pulmonary delivery of the [ETH:Booster] pair in TB chemotherapy.

Lentiviral vectors are among the most effective viral vectors for vaccination. In clear contrast to the reference adenoviral vectors, lentiviral vectors have a high potential for transducing dendritic cells in vivo. Within these cells, which are the most efficient at activating naive T cells, lentiviral vectors induce endogenous expression of transgenic antigens that directly access antigen presentation pathways without the need for external antigen capture or cross-presentation. Lentiviral vectors induce strong, robust, and long-lasting humoral, CD8+ T-cell immunity and effective protection against several infectious diseases. There is no pre-existing immunity to lentiviral vectors in the human population and the very low pro-inflammatory properties of these vectors pave the way for their use in mucosal vaccination. In this review, we have mainly summarized the immunological aspects of lentiviral vectors, their recent optimization to induce CD4+ T cells, and our recent data on lentiviral vector-based vaccination in preclinical models, including prophylaxis against flaviviruses, SARS-CoV-2, and Mycobacterium tuberculosis.

Dengue virus (DENV) is responsible for approximately 100 million cases of dengue fever annually, including severe forms such as hemorrhagic dengue and dengue shock syndrome. Despite intensive vaccine research and development spanning several decades, a universally accepted and approved vaccine against dengue fever has not yet been developed. The major challenge associated with the development of such a vaccine is that it should induce simultaneous and equal protection against the four DENV serotypes, because past infection with one serotype may greatly increase the severity of secondary infection with a distinct serotype, a phenomenon known as antibody-dependent enhancement (ADE). Using a lentiviral vector platform that is particularly suitable for the induction of cellular immune responses, we designed a tetravalent T-cell vaccine candidate against DENV (“LV-DEN”). This vaccine candidate has a strong CD8 + T-cell immunogenicity against the targeted non-structural DENV proteins, without inducing antibody response against surface antigens. Evaluation of its protective potential in the preclinical flavivirus infection model, i.e., mice knockout for the receptor to the type I IFN, demonstrated its significant protective effect against four distinct DENV serotypes, based on reduced weight loss, viremia, and viral loads in peripheral organs of the challenged mice. These results provide proof of concept for the use of lentiviral vectors for the development of efficient polyvalent T-cell vaccine candidates against all DENV serotypes.

We report a lentiviral vector harboring the human β2-microglobulin promoter, with predominant expression in immune cells and minimal proximal enhancers to improve vector safety. This lentiviral vector efficiently transduces major dendritic cell subsets in vivo. With a mycobacterial immunogen, we observed distinct functional signatures and memory phenotype in lentiviral vector- or Adenovirus type 5 (Ad5)-immunized mice, despite comparable antigen-specific CD8+ T cell magnitudes. Compared to Ad5, lentiviral vector immunization resulted in higher multifunctional and IL-2-producing CD8+ T cells. Furthermore, lentiviral vector immunization primed CD8+ T cells towards central memory phenotype, while Ad5 immunization favored effector memory phenotype. Studies using HIV antigens in outbred rats demonstrated additional clear-cut evidence for an immunogenic advantage of lentiviral vector over Ad5. Additionally, lentiviral vector provided enhance therapeutic anti-tumor protection than Ad5. In conclusion, coupling lentiviral vector with β2-microglobulin promoter represents a promising approach to produce long-lasting, high-quality cellular immunity for vaccinal purposes.Ku et al. report a lentiviral (LV) vector system in which expression of vaccine antigens is driven by β2-microglobulin (β2m) promoter. Conducting comparative studies in mice and rats, they find that LV-based vaccine outperforms the ‘gold-standard’ Ad5 by inducing a polyfunctional and long-lived immune response.

COVID‐19 vaccines already in use or in clinical development may have reduced efficacy against emerging SARS‐CoV‐2 variants. In addition, although the neurotropism of SARS‐CoV‐2 is well established, the vaccine strategies currently developed have not taken into account protection of the central nervous system. Here, we generated a transgenic mouse strain expressing the human angiotensin‐converting enzyme 2, and displaying unprecedented brain permissiveness to SARS‐CoV‐2 replication, in addition to high permissiveness levels in the lung. Using this stringent transgenic model, we demonstrated that a non‐integrative lentiviral vector, encoding for the spike glycoprotein of the ancestral SARS‐CoV‐2, used in intramuscular prime and intranasal boost elicits sterilizing protection of lung and brain against both the ancestral virus, and the Gamma (P.1) variant of concern, which carries multiple vaccine escape mutations. Beyond induction of strong neutralizing antibodies, the mechanism underlying this broad protection spectrum involves a robust protective T‐cell immunity, unaffected by the recent mutations accumulated in the emerging SARS‐CoV‐2 variants. SYNOPSIS A lentiviral vector‐based vaccine, used in prime and intranasal boost, induces strong B‐ and T‐cell immunity. This vaccine candidate protects against both pulmonary and neurological COVID‐19 caused by either ancestral or emerging SARS‐CoV‐2. A new human ACE2 transgenic mouse strain provides a model to assess the efficiency of vaccine candidates against the neurological COVID‐19. A non‐integrative lentiviral vector, encoding for the ancestral SARS‐CoV‐2 Spike (LV::S), used in prime and intranasal boost, elicits strong protection of brain. LV::S induces full cross‐protection of lung and brain against Gamma (P.1) variant of concern, which carries multiple vaccine escape mutations. LV::S induces strong protective T‐cell immunity, unaffected by the recent mutations accumulated in the emerging SARS‐CoV‐2 variants. Graphical Abstract A lentiviral vector‐based vaccine, used in prime and intranasal boost, induces strong B‐ and T‐cell immunity. This vaccine candidate protects against both pulmonary and neurological COVID‐19 caused by either ancestral or emerging SARS‐CoV‐2.

Reconstructing the evolutionary origins of Mycobacterium tuberculosis , the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M . tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens , M . spongiae is the most closely related species to the M . tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M . tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M . tuberculosis , together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M . tuberculosis challenge compared to BCG vaccination alone. The discovery of M . spongiae adds to our understanding of the emergence of the M . tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M . tuberculosis .

Tuberculosis is the deadliest infectious disease worldwide. Although the BCG vaccine is widely used, it does not efficiently protect against pulmonary tuberculosis and an improved tuberculosis vaccine is therefore urgently needed. Mycobacterium tuberculosis uses different ESX/Type VII secretion (T7S) systems to transport proteins important for virulence and host immune responses. We recently reported that secretion of T7S substrates belonging to the mycobacteria-specific Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins of the PGRS (polymorphic GC-rich sequences) and MPTR (major polymorphic tandem repeat) subfamilies required both a functional ESX-5 system and a functional PPE38/71 protein for secretion. Inactivation of ppe38/71 and the resulting loss of PE_PGRS/PPE-MPTR secretion were linked to increased virulence of M. tuberculosis strains. Here, we show that a predicted total of 89 PE_PGRS/PPE-MPTR surface proteins are not exported by certain animal-adapted strains of the M. tuberculosis complex including M. bovis. This Δppe38/71-associated secretion defect therefore also occurs in the M. bovis-derived tuberculosis vaccine BCG and could be partially restored by introduction of the M. tuberculosis ppe38-locus. Epitope mapping of the PPE-MPTR protein PPE10, further allowed us to monitor T-cell responses in splenocytes from BCG/M. tuberculosis immunized mice, confirming the dependence of PPE10-specific immune-induction on ESX-5/PPE38-mediated secretion. Restoration of PE_PGRS/PPE-MPTR secretion in recombinant BCG neither altered global antigenic presentation or activation of innate immune cells, nor protective efficacy in two different mouse vaccination-infection models. This unexpected finding stimulates a reassessment of the immunomodulatory properties of PE_PGRS/PPE-MPTR proteins, some of which are contained in vaccine formulations currently in clinical evaluation.