Catharine M. Bosio

Efficient immunization and cross-priming by vaccine adjuvants containing TLR3 or TLR9 agonists complexed to cationic liposomes.

Complexing TLR9 agonists such as plasmid DNA to cationic liposomes markedly potentiates their ability to activate innate immunity. We therefore reasoned that liposomes complexed with DNA or other TLR agonists could be used as effective vaccine adjuvants. To test this hypothesis, the vaccine adjuvant effects of liposomes complexed to TLR agonists were assessed in mice. We found that liposomes complexed to nucleic acids (liposome-Ag-nucleic acid complexes; LANAC) were particularly effective adjuvants for eliciting CD4+ and CD8+ T cell responses against peptide and protein Ags. Notably, LANAC containing TLR3 or TLR9 agonists effectively cross-primed CD8+ T cell responses against even low doses of protein Ags, and this effect was independent of CD4+ T cell help. Ag-specific CD8+ T cells elicited by LANAC adjuvants were functionally active and persisted for long periods of time in tissues. In a therapeutic tumor vaccine model, immunization with the melanoma peptide trp2 and LANAC adjuvant controlled the growth of established B16 melanoma tumors. In a prophylactic vaccine model, immunization with the Mycobacterium tuberculosis protein ESAT-6 with LANAC adjuvant elicited significant protective immunity against aerosol challenge with virulent M. tuberculosis. These results suggest that certain TLR agonists can be combined with cationic liposomes to produce uniquely effective vaccine adjuvants capable of eliciting strong T cell responses against protein and peptide Ags.

Active Suppression of the Pulmonary Immune Response by Francisella tularensis Schu4

Francisella tularensis is an obligate, intracellular bacterium that causes acute, lethal disease following inhalation. As an intracellular pathogen F. tularensis must invade cells, replicate, and disseminate while evading host immune responses. The mechanisms by which virulent type A strains of Francisella tularensis accomplish this evasion are not understood. Francisella tularensis has been shown to target multiple cell types in the lung following aerosol infection, including dendritic cells (DC) and macrophages. We demonstrate here that one mechanism used by a virulent type A strain of F. tularensis (Schu4) to evade early detection is by the induction of overwhelming immunosuppression at the site of infection, the lung. Following infection and replication in multiple pulmonary cell types, Schu4 failed to induce the production of proinflammatory cytokines or increase the expression of MHCII or CD86 on the surface of resident DC within the first few days of disease. However, Schu4 did induce early and transient production of TGF-β, a potent immunosuppressive cytokine. The absence of DC activation following infection could not be attributed to the apoptosis of pulmonary cells, because there were minimal differences in either annexin or cleaved caspase-3 staining in infected mice compared with that in uninfected controls. Rather, we demonstrate that Schu4 actively suppressed in vivo responses to secondary stimuli (LPS), e.g., failure to recruit granulocytes/monocytes and stimulate resident DC. Thus, unlike attenuated strains of F. tularensis, Schu4 induced broad immunosuppression within the first few days after aerosol infection. This difference may explain the increased virulence of type A strains compared with their more attenuated counterparts.

Francisella tularensis Induces Aberrant Activation of Pulmonary Dendritic Cells

Francisella tularensis is an obligate intracellular bacterium that induces severe, acute, often fatal disease when acquired by the respiratory route. Despite the seriousness of this pathogen, very little is understood about its interaction with key target cells in the airways and lungs (alveolar macrophages and airway dendritic cells (DC)) after inhalation. In this study we demonstrate replication of F. tularensis in primary DC. Early after infection, F. tularensis induced increased expression of MHC class II and CD86 on DC, but not macrophages. This was followed by depletion of DC from the airways and lungs. Despite logarithmic replication and phenotypic maturation of DC, F. tularensis failed to induce production of several key proinflammatory cytokines, including TNF-α and IL-6, from DC. However, F. tularensis infection did elicit production of the potent immunosuppressive cytokine, TGF-β. Furthermore, F. tularensis actively suppressed the ability of DC to secrete cytokines in response to specific TLR agonists. Finally, we also found that infection of DC and macrophages in the lungs appears to actually increase the severity of pulmonary infection with F. tularensis. For example, depletion of airway DC and alveolar macrophages before infection resulted in significantly prolonged survival times. Together, these data suggest F. tularensis is able to selectively uncouple Ag-presenting functions from proinflammatory cytokine secretion by critical APCs in the lungs, which may serve to create a relatively immunosuppressive environment favorable to replication and dissemination of the organism.

Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophages

The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis ssp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages, to characterize its intracellular biology and identify pathogenic determinants based on their intracellular expression profiles. Phagocytosed bacteria rapidly responded to their intracellular environment and subsequently altered their transcriptional profile. Differential gene expression profiles were revealed that correlated with specific intracellular locale of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of transport and metabolic genes characterized the cytosolic replication stage. Expression of the Francisella Pathogenicity Island (FPI) genes, which are required for intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci encoding putative hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated. Among these, deletion of FTT0383, FTT0369c or FTT1676 abolished the ability of Schu S4 to survive or proliferate intracellularly and cause lethality in mice, therefore identifying novel determinants of Francisella virulence from their intracellular expression profile.

Lung environment determines unique phenotype of alveolar macrophages

Alveolar macrophages (AM) are the most abundant antigen-presenting cells in the lungs, and they play a critical role in regulating pulmonary immune responses to inhaled pathogens and to allergens. However, compared with macrophages in other body sites, AM have an unusual phenotype that, in many respects, resembles the phenotype of dendritic cells (DC). Therefore, to more fully define the unique nature of AM, we compared the phenotype and function of AM with the phenotype and function of resident peritoneal lavage-derived macrophages (PLM). We found striking phenotypic differences between AM and PLM, particularly with regard to CD11c expression, and we also observed that AM had a significantly better antigen-presenting capability than PLM. Therefore, we investigated the role of the local airway environment in generation of the unusual phenotype of AM. We carried out cell transfer experiments to compare macrophage differentiation in the airways with that in the peritoneal cavity. We observed significant upregulation of CD11c expression on bone marrow macrophages and peritoneal macrophages when they were adoptively transferred into the airways. In contrast, CD11c expression was not upregulated after cell transfer into the peritoneal cavity, whereas CD11b expression was significantly increased. In vitro, culture of bone marrow-adherent cells with surfactant protein D (SP-D) or granulocyte/macrophage colony-stimulating factor (GM-CSF) induced significant upregulation of CD11c expression, and in vivo GM-CSF concentrations were significantly higher in bronchoalveolar than in peritoneal lavage fluid. Finally, GM-CSF(-/-) mice failed to develop CD11c(+) AM, but CD11c(+) AM were present in SP-D(-/-) mice. However, macrophages from GM-CSF(-/-) bone marrow could upregulate CD11c expression when transferred to the airways of wild-type mice. These results suggest that the airway environment promotes development of macrophages with unique DC-like characteristics and that this unusual phenotype is determined, to a large degree, by locally high concentrations of GM-CSF and, possibly, SP-D.

Susceptibility to Secondary Francisella tularensis Live Vaccine Strain Infection in B-Cell-Deficient Mice Is Associated with Neutrophilia but Not with Defects in Specific T-Cell-Mediated Immunity

ABSTRACT Previous studies have demonstrated a role for B cells, not associated with antibody production, in protection against lethal secondary infection of mice with Francisella tularensislive vaccine strain (LVS). However, the mechanism by which B cells contribute to this protection is not known. To study the specific role of B cells during secondary LVS infection, we developed an in vitro culture system that mimics many of the same characteristics of in vivo infection. Using this culture system, we showed that B cells do not directly control LVS infection but that control of LVS growth is mediated primarily by LVS-primed T cells. Importantly, B cells were not required for the generation of effective memory T cells since LVS-primed, B-cell-deficient (BKO) mice generated CD4+ and CD8+ T cells that controlled LVS infection similarly to LVS-primed CD4+ and CD8+ T cells from wild-type mice. The control of LVS growth appeared to depend primarily on gamma interferon and nitric oxide and was similar in wild-type and BKO mice. Rather, the inability of BKO mice to survive secondary LVS infection was associated with marked neutrophil influx into the spleen very early after challenge. The neutrophilia was directly associated with B cells, since BKO mice reconstituted with naive B cells prior to a secondary challenge with LVS had decreased bacterial loads and neutrophils in the spleen and survived.

Direct and indirect impairment of human dendritic cell function by virulent Francisella tularensis Schu S4.

ABSTRACT The gram-negative, facultative intracellular bacterium Francisella tularensis causes acute, lethal pneumonic disease following infection with only 10 CFU. The mechanisms used by the bacterium to accomplish this in humans are unknown. Here, we demonstrate that virulent, type A F. tularensis strain Schu S4 efficiently infects and replicates in human myeloid dendritic cells (DCs). Despite exponential replication over time, Schu S4 failed to stimulate transforming growth factor β, interleukin-10 (IL-10), IL-6, IL-1β, IL-12, tumor necrosis factor alpha, alpha interferon (IFN-α), and IFN-β throughout the course of infection. Schu S4 also suppressed the ability of directly infected DCs to respond to different Toll-like receptor agonists. Furthermore, we also observed functional inhibition of uninfected bystander cells. This inhibition was mediated, in part, by a heat-stable bacterial component. Lipopolysaccharide (LPS) from Schu S4 was present in Schu S4-conditioned medium. However, Schu S4 LPS was weakly inflammatory and failed to induce suppression of DCs at concentrations below 10 μg/ml, and depletion of Schu S4 LPS did not significantly alleviate the inhibitory effect of Schu S4-conditioned medium in uninfected human DCs. Together, these data show that type A F. tularensis interferes with the ability of a central cell type of the immune system, DCs, to alert the host of infection both intra- and extracellularly. This suggests that immune dysregulation by F. tularensis operates on a broader and more comprehensive scale than previously appreciated.

Oral Vaccination with Salmonella Simultaneously Expressing Yersinia pestis F1 and V Antigens Protects against Bubonic and Pneumonic Plague

The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against ∼1000 LD50 Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD50 Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.

A Novel Role for Plasmin-Mediated Degradation of Opsonizing Antibody in the Evasion of Host Immunity by Virulent, but Not Attenuated, Francisella tularensis

Opsonization by Abs represents a critical component of the host immune response against many pathogens. The mechanisms by which virulent microbes evade this protective response are not completely understood. In disease mediated by Francisella tularensis, Ab can effectively protect against infections with attenuated strains, for example, LVS, but not virulent strains such as SchuS4. Thus, it is likely that SchuS4 has mechanisms, which are not present in LVS, that allow evasion of opsonization by Ab, dampening the protective effects of these host molecules. Here we demonstrate that evasion of Ab-mediated opsonization and phagocytosis by the highly virulent SchuS4 is associated with its ability to bind the host serine protease plasmin. SchuS4, but not the closely related LVS, bound active plasmin. Plasmin bound SchuS4 degraded exogenous and opsonizing Abs, whereas LVS failed to do so. Furthermore, plasmin-mediated inhibition of Ab opsonization by SchuS4 also inhibited Ab-mediated uptake of this bacterium by macrophages. Ab-mediated uptake of uncoated and opsonized SchuS4 elicited a strong proinflammatory response in infected macrophages. However, plasmin-coated, opsonized SchuS4 poorly elicited production of these protective proinflammatory cytokines. This unique host-pathogen interplay is a novel immune evasion strategy utilized by virulent F. tularensis, and it provides one explanation for the ability of Ab to protect against attenuated, but not virulent, strains of F. tularensis. This mechanism may also represent a more common hereto unrecognized strategy by which virulent bacteria evade detection and clearance by Ig.

NKp30-dependent cytolysis of filovirus-infected human dendritic cells

Understanding how protective innate immune responses are generated is crucial to defeating highly lethal emerging pathogens. Accumulating evidence suggests that potent innate immune responses are tightly linked to control of Ebola and Marburg filoviral infections. Here, we report that unlike authentic or inactivated Ebola and Marburg, filovirus‐derived virus‐like particles directly activated human natural killer (NK) cells in vitro, evidenced by pro‐inflammatory cytokine production and enhanced cytolysis of permissive target cells. Further, we observed perforin‐ and CD95L‐mediated cytolysis of filovirus‐infected human dendritic cells (DCs), primary targets of filovirus infection, by autologous NK cells. Gene expression knock‐down studies directly linked NK cell lysis of infected DCs to upregulation of the natural cytotoxicity receptor, NKp30. These results are the first to propose a role for NK cells in the clearance of infected DCs and the potential involvement of NKp30‐mediated cytolysis in control of viral infection in vivo. Further elucidation of the biology of NK cell activation, specifically natural cytotoxicity receptors like NKp30 and NKp46, promises to aid our understanding of microbial pathology.