Our previous studies demonstrated that Mycobacterium bovis bacillus Calmette-Guérin (BCG) can directly interact with human NK cells and induce the proliferation, gamma interferon production, and cytotoxic activity of such cells without the need for accessory cells. Thus, the aim of the present study was to identify the putative receptor(s) responsible for the recognition of BCG by human NK cells and potentially involved in the activation of NK cells. To this end, we first investigated the surface expression of three NK cell-activating receptors belonging to the natural cytoxicity receptor (NCR) family on highly purified human NK cells upon in vitro direct stimulation with BCG. An induction of the surface expression of NKp44, but not of NKp30 or NKp46, was observed after 3 and 4 days of in vitro stimulation with live BCG. The NKp44 induction involved mainly a particular NK cell subset expressing the CD56 marker at high density, CD56 ^bright . In order to establish whether NKp44 could directly bind to BCG, whole BCG cells were stained with soluble forms of the three NCRs chimeric for the human immunoglobulin G (IgG) Fc fragment (NKp30-Fc, NKp44-Fc, NKp46-Fc), followed by incubation with a phycoerythrin (PE)-conjugated goat anti-human IgG antibody. Analysis by flow cytometry of the complexes revealed a higher PE fluorescence intensity for BCG incubated with NKp44-Fc than for BCG incubated with NKp30-Fc, NKp46-Fc, or negative controls. The binding of NKp44-Fc to the BCG surface was confirmed with immunogold labeling using transmission electron microscopy, suggesting the presence of a putative ligand(s) for human NKp44 on the BCG cell wall. Similar binding assays performed on a number of gram-positive and gram-negative bacteria revealed a pattern of NKp44-Fc binding restricted to members of the genus Mycobacterium , to the mycobacterium-related species Nocardia farcinica , and to Pseudomonas aeruginosa . Altogether, the results obtained indicate, for the first time, that at least one member of the NCR family (NKp44) may be involved in the direct recognition of bacterial pathogens by human NK cells.

We have previously demonstrated that a soluble form of the human NK cell natural cytotoxicity receptor NKp44, binds to the surface of Mycobacterium tuberculosis (MTB). Herein, we investigated the interaction of MTB cell wall components (CWC) with NKp44 or with Toll‐like receptor 2 (TLR2) and the role of NKp44 and TLR2 in the direct activation of NK cells upon stimulation with MTB CWC. By using several purified bacterial CWC in an ELISA, we demonstrated that NKp44 was able to bind to the MTB cell wall core mycolyl‐arabinogalactan‐peptidoglycan (mAGP) as well as to mycolic acids (MA) and arabinogalactan (AG), while soluble TLR2 bound to MTB peptidoglycan (PG), but not to MA or AG. The mAGP complex induced NK cell expression of CD25, CD69, NKp44 and IFN‐γ production at levels comparable to M. bovis Bacillus Calmette–Guérin‐stimulated (BCG) cells. While AG and MA used alone failed to induce NK cell activation, mycobacterial PG‐exhibited NK cell stimulatory capacity. Activation of resting NK cells by mAGP and IFN‐γ production were inhibited by anti‐TLR2 MAb, but not by anti‐NKp44 MAb. Differently, anti‐NKp44 MAb partially inhibited CD69 expression on NK cells pre‐activated with IL‐2 and then stimulated with mAGP or whole BCG. Overall, these results provide evidence that components abundant in mycobacterial cell wall are able to interact with NKp44 (AG, MA) and TLR‐2 (PG), respectively. While interaction of TLR2 with mycobacterial cell wall promotes activation of resting NK cells and IFN‐γ production, NKp44 interaction with its putative ligands could play a secondary role in maintaining cell activation.

The recently described ESX-5 secretion system of Mycobacterium tuberculosis is one of the most important modulators of host-pathogen interactions due to its crucial impact on PPE protein secretion, cell wall stability and virulence. Although various components of the ESX-5 secretion machinery have been defined, other ESX-5 core components still remain to be characterized. In this study, we focused on EccB₅ and EccC₅, a transmembrane protein (EccB₅) and a membrane-bound ATPase (EccC₅), both predicted to be building blocks of the M. tuberculosis ESX-5 membrane-associated complex. In vitro expression studies demonstrated that EccB₅ and EccC₅ encoding genes constitute an operon. The expression of this operon is essential for M. tuberculosis, since the deletion of the eccB₅-eccC₅ genomic segment at the ESX-5 locus is possible only after the integration of a second functional copy of eccB₅-eccC₅ genes into the M. tuberculosis chromosome. The characterization of two M. tuberculosis conditional mutant strains (Mtb_PptreccB₅ and Mtb_PptreccC₅), in which the eccB₅-eccC₅ operon or the eccC₅ gene, respectively, were expressed under the control of an anhydrotetracycline-repressible promoter, confirmed that the repression of eccB₅-eccC₅ genes is detrimental for growth of M. tuberculosis both in vitro and in THP-1 human macrophage cell line. Moreover, analysis of the secretome of Mtb_PptreccB₅-eccC₅ and Mtb_PptreccC₅ strains revealed that both EccB₅ and EccC₅ are required for secretion of ESX-5 specific substrates, thus confirming that they are indeed components of the ESX-5 secretion machinery. Taken together these findings demonstrate the importance of an intact and functional ESX-5 system for viability of M. tuberculosis, thus opening new interesting options for alternative antimycobacterial control strategies.

The development of effective vaccines against bacterial lung infections requires the induction of protective, pathogen-specific immune responses without deleterious inflammation within the pulmonary environment. Here, we made use of a polysaccharide-adjuvanted vaccine approach to elicit resident pulmonary T cells to protect against aerosol Mycobacterium tuberculosis infection. Intratracheal administration of the multistage fusion protein CysVac2 and the delta-inulin adjuvant Advax™ (formulated with a TLR9 agonist) provided superior protection against aerosol M. tuberculosis infection in mice, compared to parenteral delivery. Surprisingly, removal of the TLR9 agonist did not impact vaccine protection despite a reduction in cytokine-secreting T cell subsets, particularly CD4⁺IFN-γ⁺IL-2⁺TNF⁺ multifunctional T cells. CysVac2/Advax-mediated protection was associated with the induction of lung-resident, antigen-specific memory CD4⁺ T cells that expressed IL-17 and RORγT, the master transcriptional regulator of Th17 differentiation. IL-17 was identified as a key mediator of vaccine efficacy, with blocking of IL-17 during M. tuberculosis challenge reducing phagocyte influx, suppressing priming of pathogen-specific CD4⁺ T cells in local lymph nodes and ablating vaccine-induced protection. These findings suggest that tuberculosis vaccines such as CysVac2/Advax that are capable of eliciting Th17 lung-resident memory T cells are promising candidates for progression to human trials.

There is an urgent need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis. Advax™ is a novel adjuvant based on delta inulin microparticles that enhances immunity with a minimal inflammatory profile and has entered human trials to protect against viral pathogens. In this report we determined if Advax displays broad applicability against important human pathogens by assessing protective immunity against infection with M. tuberculosis. The fusion protein CysVac2, comprising the M. tuberculosis antigens Ag85B (Rv1886c) and CysD (Rv1285) formulated with Advax provided significant protection in the lungs of M. tuberculosis-infected mice. Protection was associated with the generation of CysVac2-specific multifunctional CD4⁺ T cells (IFN-γ⁺TNF⁺IL-2⁺). Addition to Advax of the TLR9 agonist, CpG oligonucleotide (Advax^CpG), improved both the immunogenicity and protective efficacy of CysVac2. Immunisation with CysVac2/Advax^CpG resulted in heightened release of the chemoattractants, CXCL1, CCL3, and TNF, and rapid influx of monocytes and neutrophils to the site of vaccination, with pronounced early priming of CysVac2-specific CD4⁺ T cells. As delta inulin adjuvants have shown an excellent safety and tolerability profile in humans, CysVac2/Advax^CpG is a strong candidate for further preclinical evaluation for progression to human trials.

Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Guérin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilised, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum. This vaccine formulation, BCG:CoVac, induced high-titre SARS-CoV-2 neutralising antibodies (NAbs) and Th1-biased cytokine release by vaccine-specific T cells, which correlated with the early emergence of T follicular helper cells in local lymph nodes and heightened levels of antigen-specific plasma B cells after vaccination. Vaccination of K18-hACE2 mice with a single dose of BCG:CoVac almost completely abrogated disease after SARS-CoV-2 challenge, with minimal inflammation and no detectable virus in the lungs of infected animals. Boosting BCG:CoVac-primed mice with a heterologous vaccine further increased SARS-CoV-2-specific antibody responses, which effectively neutralised B.1.1.7 and B.1.351 SARS-CoV-2 variants of concern. These findings demonstrate the potential for BCG-based vaccination to protect against major SARS-CoV-2 variants circulating globally.