Claude Leclerc

Neonatal adaptive immunity comes of age

The past decade has brought great strides in our understanding of adaptive immunity in neonatal mice. Although poor immune responses are commonly observed, it is now clear that mature function can be achieved by all arms of the adaptive immune system. An ever-increasing body of evidence indicates that the neonatal period of life is a unique developmental stage in which responses are highly plastic and dependent on the conditions of antigen exposure. This review focuses on our new understanding in mice and, where it is clear that related phenomena occur, in humans.

Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis

The live tuberculosis vaccines Mycobacterium bovis BCG (bacille Calmette-Guérin) and Mycobacterium microti both lack the potent, secreted T-cell antigens ESAT-6 (6-kDa early secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein). This is a result of independent deletions in the region of deletion-1 (RD1) locus, which is intact in virulent members of the Mycobacterium tuberculosis complex. To increase their immunogenicity and protective capacity, we complemented both vaccines with different constructs containing the esxA and esxB genes, which encode ESAT-6 and CFP-10 respectively, as well as a variable number of flanking genes. Only reintroduction of the complete locus, comprising at least 11 genes, led to full secretion of the antigens and resulted in specific ESAT-6–dependent immune responses; this suggests that the flanking genes encode a secretory apparatus. Mice and guinea pigs vaccinated with the recombinant strain BCG::RD1-2F9 were better protected against challenge with M. tuberculosis, showing less severe pathology and reduced dissemination of the pathogen, as compared with control animals immunized with BCG alone.

Dissection of ESAT-6 System 1 of Mycobacterium tuberculosis and Impact on Immunogenicity and Virulence

ABSTRACT The dedicated secretion system ESX-1 of Mycobacterium tuberculosis encoded by the extended RD1 region (extRD1) assures export of the ESAT-6 protein and its partner, the 10-kDa culture filtrate protein CFP-10, and is missing from the vaccine strains M. bovis BCG and M. microti. Here, we systematically investigated the involvement of each individual ESX-1 gene in the secretion of both antigens, specific immunogenicity, and virulence. ESX-1-complemented BCG and M. microti strains were more efficiently engulfed by bone-marrow-derived macrophages than controls, and this may account for the enhanced in vivo growth of ESX-1-carrying strains. Inactivation of gene pe35 (Rv3872) impaired expression of CFP-10 and ESAT-6, suggesting a role in regulation. Genes Rv3868, Rv3869, Rv3870, Rv3871, and Rv3877 encoding an ATP-dependent chaperone and translocon were essential for secretion of ESAT-6 and CFP-10 in contrast to ppe68 Rv3873 and Rv3876, whose inactivation did not impair secretion of ESAT-6. A strict correlation was found between ESAT-6 export and the generation of ESAT-6 specific T-cell responses in mice. Furthermore, ESAT-6 secretion and specific immunogenicity were almost always correlated with enhanced virulence in the SCID mouse model. Only loss of Rv3865 and part of Rv3866 did not affect ESAT-6 secretion or immunogenicity but led to attenuation. This suggests that Rv3865/66 represent a new virulence factor that is independent from ESAT-6 secretion. The present study has allowed us to identify new aspects of the extRD1 region of M. tuberculosis and to explore its role in the pathogenesis of tuberculosis.

Coactivation of Syk Kinase and MyD88 Adaptor Protein Pathways by Bacteria Promotes Regulatory Properties of Neutrophils

Neutrophils are one of the first lines of defense against microbial pathogens and are rapidly recruited at the infection site upon inflammatory conditions. We show here that after bacterial stimulation, and in contrast to monocytes and macrophages, murine neutrophils contributed poorly to inflammatory responses; however, they secreted high amounts of the anti-inflammatory cytokine IL-10 in a DAP12 adaptor-Syk kinase and MyD88 adaptor-dependent manner. Cotriggering of TLR-MyD88- and C-type lectin receptor (CLR)-Syk-dependent pathways led to a quick and sustained phosphorylation of p38 MAP and Akt kinases in neutrophils. In vivo, both Gram-negative bacteria and mycobacteria induced the recruitment of neutrophils secreting IL-10. In acute mycobacterial infection, neutrophil-derived IL-10 controlled the inflammatory response of dendritic cells, monocytes and macrophages in the lung. During a chronic infection, neutrophil depletion promoted inflammation and decreased the mycobacterial burden. Therefore, neutrophils can have a previously unsuspected regulatory role during acute and chronic microbial infections.

Dendritic Cells Are Host Cells for Mycobacteria In Vivo That Trigger Innate and Acquired Immunity

In the present study, we investigated in vivo the infection and APC functions of dendritic cells (DC) and macrophages (Mφ) after administration of live mycobacteria to mice. Experiments were conducted with Mycobacterium bovis bacillus Calmette-Guerin (BCG) or a rBCG expressing a reporter Ag. Following infection of mice, DC and Mφ were purified and the presence of immunogenic peptide/MHC class II complexes was detected ex vivo on sorted cells, as was the secretion of IL-12 p40. We show in this study that DC is a host cell for mycobacteria, and we provide an in vivo detailed picture of the role of Mφ and DC in the mobilization of immunity during the early stages of a bacterial infection. Strikingly, BCG bacilli survive but remain stable in number in the DC leukocyte subset during the first 2 wk of infection. As Ag presentation by DC is rapidly lost, this suggests that DC may represent a hidden reservoir for mycobacteria.

Control of M. tuberculosis ESAT-6 Secretion and Specific T Cell Recognition by PhoP

Analysis of mycobacterial strains that have lost their ability to cause disease is a powerful approach to identify yet unknown virulence determinants and pathways involved in tuberculosis pathogenesis. Two of the most widely used attenuated strains in the history of tuberculosis research are Mycobacterium bovis BCG (BCG) and Mycobacterium tuberculosis H37Ra (H37Ra), which both lost their virulence during in vitro serial passage. Whereas the attenuation of BCG is due mainly to loss of the ESAT-6 secretion system, ESX-1, the reason why H37Ra is attenuated remained unknown. However, here we show that a point mutation (S219L) in the predicted DNA binding region of the regulator PhoP is involved in the attenuation of H37Ra via a mechanism that impacts on the secretion of the major T cell antigen ESAT-6. Only H37Ra “knock-ins” that carried an integrated cosmid with the wild-type phoP gene from M. tuberculosis H37Rv showed changes in colony morphology, increased virulence, ESAT-6 secretion, and induction of specific T cell responses, whereas other H37Ra constructs did not. This finding established a link between the PhoP regulator and ESAT-6 secretion that opens exciting new perspectives for elucidating virulence regulation in M. tuberculosis.

Genomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of Mycobacterium tuberculosis

Global spread and limited genetic variation are hallmarks of M. tuberculosis, the agent of human tuberculosis. In contrast, Mycobacterium canettii and related tubercle bacilli that also cause human tuberculosis and exhibit unusual smooth colony morphology are restricted to East Africa. Here, we sequenced and analyzed the whole genomes of five representative strains of smooth tubercle bacilli (STB) using Sanger (4–5× coverage), 454/Roche (13–18× coverage) and/or Illumina DNA sequencing (45–105× coverage). We show that STB isolates are highly recombinogenic and evolutionarily early branching, with larger genome sizes, higher rates of genetic variation, fewer molecular scars and distinct CRISPR-Cas systems relative to M. tuberculosis. Despite the differences, all tuberculosis-causing mycobacteria share a highly conserved core genome. Mouse infection experiments showed that STB strains are less persistent and virulent than M. tuberculosis. We conclude that M. tuberculosis emerged from an ancestral STB-like pool of mycobacteria by gain of persistence and virulence mechanisms, and we provide insights into the molecular events involved.

Insect Baculoviruses Strongly Potentiate Adaptive Immune Responses by Inducing Type I IFN

Baculoviruses (BVs) are dsDNA viruses that are pathogenic for insects. They have been used worldwide as selective bioinsecticides and for producing recombinant proteins in insect cells. Surprisingly, despite their widespread use in research and industry and their dissemination in the environment, the potential effects of these insect viruses on the immune responses of mammals remain totally unknown. We show in this study that BVs have strong adjuvant properties in mice, promoting potent humoral and CD8+ T cell adaptive responses against coadministered Ag. BVs also induce the in vivo maturation of dendritic cells and the production of inflammatory cytokines. We demonstrate that BVs play a major role in the strong immunogenicity of virus-like particles produced in the BV-insect cell expression system. The presence of even small numbers of BVs among the recombinant proteins produced in the BV expression system may therefore strengthen the immunological properties of these proteins. This adjuvant behavior of BVs is mediated primarily by IFN-αβ, although mechanisms independent of type I IFN signaling are also involved. This study demonstrates that nonpathogenic insect viruses may have a strong effect on the mammalian immune system.

Genome analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of the etiologic agent of tuberculosis

Global spread and limited genetic variation are hallmarks of M. tuberculosis, the agent of human tuberculosis. In contrast, Mycobacterium canettii and related tubercle bacilli that also cause human tuberculosis and exhibit unusual smooth colony morphology are restricted to East Africa. Here, we sequenced and analyzed the whole genomes of five representative strains of smooth tubercle bacilli (STB) using Sanger (4–5× coverage), 454/Roche (13–18× coverage) and/or Illumina DNA sequencing (45–105× coverage). We show that STB isolates are highly recombinogenic and evolutionarily early branching, with larger genome sizes, higher rates of genetic variation, fewer molecular scars and distinct CRISPR-Cas systems relative to M. tuberculosis. Despite the differences, all tuberculosis-causing mycobacteria share a highly conserved core genome. Mouse infection experiments showed that STB strains are less persistent and virulent than M. tuberculosis. We conclude that M. tuberculosis emerged from an ancestral STB-like pool of mycobacteria by gain of persistence and virulence mechanisms, and we provide insights into the molecular events involved.

CD8α2 CD11b+ Dendritic Cells Present Exogenous Virus-like Particles to CD8+ T Cells and Subsequently Express CD8α and CD205 Molecules

Recombinant porcine parvovirus virus-like particles (PPV-VLPs) are particulate exogenous antigens that induce a strong, specific cytotoxic T lymphocyte (CTL) response in the absence of adjuvant. In the present report, we demonstrate in vivo that dendritic cells (DCs) present PPV-VLPs to CD8+ T cells after intracellular processing. PPV-VLPs are captured by DCs with a high efficacy, which results in the delivery of these exogenous antigens to 50% of the whole spleen DC population. In vivo, a few hours after injection, PPV-VLPs are presented exclusively to CD8+ T cells by CD8α− DCs, whereas 15 hours later they are presented mainly by CD8α+ DCs. After PPV-VLPs processing, a fraction of CD11b+ DCs undergo phenotypic changes, i.e., the up-regulation of CD8α and CD205 and the loss of CD4 molecules on their surface. The failure to detect mRNA coding for CD8α in CD11b+ DCs suggests that CD8α expression by these cells is not due to de novo synthesis. In recombination-activating gene knockout mice (Rag−/−), CD11b+ DCs did not express CD8α and PPV-VLPs presentation by CD8α+ DCs was severely diminished. These results indicate that both CD8α− and CD8α+ DCs play an important role in the induction of CTL responses by exogenous antigens, such as VLP.