Daniela Damjanovic

Influenza infection leads to increased susceptibility to subsequent bacterial superinfection by impairing NK cell responses in the lung.

Influenza viral infection is well-known to predispose to subsequent bacterial superinfection in the lung but the mechanisms have remained poorly defined. We have established a murine model of heterologous infections by an H1N1 influenza virus and Staphylococcus aureus. We found that indeed prior influenza infection markedly increased the susceptibility of mice to secondary S. aureus superinfection. Severe sickness and heightened bacterial infection in flu and S. aureus dual-infected animals were associated with severe immunopathology in the lung. We further found that flu-experienced lungs had an impaired NK cell response in the airway to subsequent S. aureus bacterial infection. Thus, adoptive transfer of naive NK cells to the airway of prior flu-infected mice restored flu-impaired antibacterial host defense. We identified that TNF-α production of NK cells played an important role in NK cell-mediated antibacterial host defense as NK cells in flu-experienced lungs had reduced TNF-α expression and adoptive transfer of TNF-α–deficient NK cells to the airway of flu-infected mice failed to restore flu-impaired antibacterial host defense. Defected NK cell function was found to be an upstream mechanism of depressed antibacterial activities by alveolar macrophages as contrast to naive wild-type NK cells, the NK cells from flu-infected or TNF-α–deficient mice failed to enhance S. aureus phagocytosis by alveolar macrophages. Together, our study identifies the weakened NK cell response in the lung to be a novel critical mechanism for flu-mediated susceptibility to bacterial superinfection.

A Human Type 5 Adenovirus–Based Tuberculosis Vaccine Induces Robust T Cell Responses in Humans Despite Preexisting Anti-Adenovirus Immunity

An adenovirus-based TB vaccine elicits T cell immunogenicity despite preexisting anti-AdHu5 immunity in people. Boosting BCG Bacille Calmette-Guérin (BCG) has long been the standard vaccine for tuberculosis (TB). However, with TB incidence on the rise, especially in communities with heavy HIV prevalence, new vaccines are needed to work with BCG to accentuate the immune response. One such way to boost a primed immune response is by delivering pathogen-specific target antigens using adenoviral vectors. Yet, which adenovirus to use remains a matter of debate: People frequently have preexisting neutralizing antibodies to the more immunogenic vectors, and rarer vectors tend to be less immunogenic. Now, Smaill et al. show that a recombinant human type 5 adenovirus (AdHu5) has safety and efficacy in both BCG− and BCG+ adults with little observed effect of preexisting neutralizing antibodies. The authors immunized healthy subjects intramuscularly with their AdHu5 vector. The immunization was safe, well tolerated, and immunogenic in both BCG− and BCG+ individuals. However, individuals previously vaccinated with BCG were more likely to have polyfunctional T cell responses, which have been associated with protection. What’s more, although the patient numbers are small, Smaill et al. did not see an appreciable effect of preexisting neutralizing antibodies on the vaccine’s ability to boost the immune response. These data suggest that, at least for certain antigens, adenovirally delivered boosts can enhance the immune response to TB. There is an urgent need to develop new tuberculosis (TB) vaccines to safely and effectively boost Bacille Calmette-Guérin (BCG)–triggered T cell immunity in humans. AdHu5Ag85A is a recombinant human type 5 adenovirus (AdHu5)–based TB vaccine with demonstrated efficacy in a number of animal species, yet it remains to be translated to human applications. In this phase 1 study, we evaluated the safety and immunogenicity of AdHu5Ag85A in both BCG-naïve and previously BCG-immunized healthy adults. Intramuscular immunization of AdHu5Ag85A was safe and well tolerated in both trial volunteer groups. Moreover, although AdHu5Ag85A was immunogenic in both trial volunteer groups, it much more potently boosted polyfunctional CD4+ and CD8+ T cell immunity in previously BCG-vaccinated volunteers. Furthermore, despite prevalent preexisting anti-AdHu5 humoral immunity in most of the trial volunteers, we found little evidence that such preexisting anti-AdHu5 immunity significantly dampened the potency of AdHu5Ag85A vaccine. This study supports further clinical investigations of the AdHu5Ag85A vaccine for human applications. It also suggests that the widely perceived negative effect of preexisting anti-AdHu5 immunity may not be universally applied to all AdHu5-based vaccines against different types of human pathogens.

Immunopathology in influenza virus infection: uncoupling the friend from foe.

Influenza epidemics and pandemics cause significant morbidity and mortality worldwide associated with severe immunopathology in the lung, and the mechanisms of such immunopathogenesis still remain poorly understood. While human studies help to understand influenza immunopathology, they provide only limited mechanistic information. On the other hand, recent studies using experimental animal models have significantly enhanced our understanding of the complex mechanisms involved in the immunopathogenesis during primary influenza or influenza-associated bacterial superinfection. This includes the involvement of acute inflammatory responses (macrophages, neutrophils, dendritic cells, toll-like receptors, cytokines, chemokines), CD4 and CD8 T cells, tissue remodeling processes, and contribution of bacterial superinfection. In particular, progress has been made in uncoupling the mechanisms that are involved in both anti-viral host defense and in immunopathogenesis from those that solely contribute to lung immunopathology. Uncoupling such events will facilitate the discovery of new intervention strategies to treat pulmonary immunopathology associated with influenza infection.

Murine airway luminal antituberculosis memory CD8 T cells by mucosal immunization are maintained via antigen-driven in situ proliferation, independent of peripheral T cell recruitment.

RATIONALE The airway luminal memory CD8 T cells induced by respiratory mucosal immunization in a murine model have been found to be critical to antituberculosis immunity. However, the mechanisms of their maintenance on airway mucosal surface still remain poorly understood. OBJECTIVES Using a model of adenovirus-based intranasal immunization we investigated the immune property and the mechanisms of maintenance of airway luminal CD8 T cells. METHODS Immune properties of airway luminal Mycobacterium tuberculosis antigen-specific CD8 T cells were examined. Proliferation of airway luminal CD8 T cells was determined by in vivo T cell-labeling techniques. The role of peripheral T cell recruitment in maintaining airway luminal CD8 T cells was investigated by blocking lymphocyte trafficking from lymphoid and peripheral tissues. The requirement of M. tuberculosis antigens for in situ T cell proliferation was evaluated using a T cell transfer approach. An airway M. tuberculosis challenge model was used to study the relationship between CD8 T cell-mediated protection and peripheral T cell recruitment. MEASUREMENTS AND MAIN RESULTS Intranasal immunization leads to elicitation of persisting M. tuberculosis antigen-specific CD8 T cells in the airway lumen, which display an activated effector memory phenotype different from those in peripheral tissues. Airway luminal T cells continuously proliferate in an antigen-dependent manner, and can be maintained even in the absence of peripheral T cell recruitment. The lungs equipped with such CD8 T cells are protected from airway M. tuberculosis challenge independent of both peripheral T cell supply and CD4 T cells. CONCLUSIONS Vaccine-inducible airway luminal antituberculosis memory CD8 T cells are self-renewable in an antigen-dependent manner, and can be maintained independent of peripheral T cell supply.

Negative Regulation of Lung Inflammation and Immunopathology by TNF-α during Acute Influenza Infection

Lung immunopathology is the main cause of influenza-mediated morbidity and death, and much of its molecular mechanisms remain unclear. Whereas tumor necrosis factor-α (TNF-α) is traditionally considered a proinflammatory cytokine, its role in influenza immunopathology is unresolved. We have investigated this issue by using a model of acute H1N1 influenza infection established in wild-type and TNF-α-deficient mice and evaluated lung viral clearance, inflammatory responses, and immunopathology. Whereas TNF-α was up-regulated in the lung after influenza infection, it was not required for normal influenza viral clearance. However, TNF-α deficiency led not only to a greater extent of illness but also to heightened lung immunopathology and tissue remodeling. The severe lung immunopathology was associated with increased inflammatory cell infiltration, anti-influenza adaptive immune responses, and expression of cytokines such as monocyte chemoattractant protein-1 (MCP-1) and fibrotic growth factor, TGF-β1. Thus, in vivo neutralization of MCP-1 markedly attenuated lung immunopathology and blunted TGF-β1 production following influenza infection in these hosts. On the other hand, in vivo transgenic expression of MCP-1 worsened lung immunopathology following influenza infection in wild-type hosts. Thus, TNF-α is dispensable for influenza clearance; however, different from the traditional belief, this cytokine is critically required for negatively regulating the extent of lung immunopathology during acute influenza infection.

CD8+ T-cell expansion and maintenance after recombinant adenovirus immunization rely upon cooperation between hematopoietic and nonhematopoietic antigen-presenting cells

We have recently reported that CD8(+) T-cell memory maintenance after immunization with recombinant human adenovirus type 5 (rHuAd5) is dependent upon persistent transgene expression beyond the peak of the response. In this report, we have further investigated the location and nature of the cell populations responsible for this sustained response. The draining lymph nodes were found to be important for primary expansion but not for memory maintenance, suggesting that antigen presentation through a nonlymphoid source was required. Using bone marrow chimeric mice, we determined that antigen presentation by nonhematopoietic antigen-presenting cells (APCs) was sufficient for maintenance of CD8(+) T-cell numbers. However, antigen presentation by this mechanism alone yielded a memory population that displayed alterations in phenotype, cytokine production and protective capacity, indicating that antigen presentation through both hematopoietic and nonhematopoietic APCs ultimately defines the memory CD8(+) T-cell response produced by rHuAd5. These results shed new light on the immunobiology of rHuAd5 vectors and provide evidence for a mechanism of CD8(+) T-cell expansion and memory maintenance that relies upon both hematopoietic and nonhematopoietic APCs.

Airway Delivery of Soluble Mycobacterial Antigens Restores Protective Mucosal Immunity by Single Intramuscular Plasmid DNA Tuberculosis Vaccination: Role of Proinflammatory Signals in the Lung

Protection by parenteral immunization with plasmid DNA vaccines against pulmonary tuberculosis (TB) is very modest. In this study, we have investigated the underlying mechanisms for the poor mucosal protective efficacy and the avenues and mechanisms to improve the efficacy of a single i.m. immunization with a monogenic plasmid DNA TB vaccine in a murine model. We show that i.m. DNA immunization fails to elicit accumulation of Ag-specific T cells in the airway lumen despite robust T cell responses in the spleen. Such systemically activated T cells cannot be rapidly mobilized into the airway lumen upon Mycobacterium tuberculosis exposure. However, airway deposition of low doses of soluble mycobacterial Ags in previously immunized mice effectively mobilizes the systemically activated T cells into the airway lumen. A fraction of such airway luminal T cells can persist in the airway lumen, undergo quick, robust expansion and activation and provide marked immune protection upon airway M. tuberculosis exposure. Airway mucosal deposition of soluble mycobacterial Ags was found to create a tissue microenvironment rich in proinflammatory molecules including chemokines and hence conducive to T cell recruitment. Thus, in vivo neutralization of MIP-1α or IFN-inducible protein-10 markedly inhibited the accumulation of Ag-specific T cells in the airway lumen. Our data suggest that immunoprotective efficacy on the mucosal surface by i.m. plasmid DNA immunization could be substantially improved by simple mucosal soluble Ag inoculation and restoration of mucosal luminal T cells. Our study holds implication for the future design of DNA vaccination strategies against intracellular infections.

Organ distribution of transgene expression following intranasal mucosal delivery of recombinant replication-defective adenovirus gene transfer vector

It is believed that respiratory mucosal immunization triggers more effective immune protection than parenteral immunization against respiratory infection caused by viruses and intracellular bacteria. Such understanding has led to the successful implementation of intranasal immunization in humans with a live cold-adapted flu virus vaccine. Furthermore there has been an interest in developing effective mucosal-deliverable genetic vaccines against other infectious diseases. However, there is a concern that intranasally delivered recombinant viral-based vaccines may disseminate to the CNS via the olfactory tissue. Initial experimental evidence suggests that intranasally delivered recombinant adenoviral gene transfer vector may transport to the olfactory bulb. However, there is a lack of quantitative studies to compare the relative amounts of transgene products in the respiratory tract, lung, olfactory bulb and brain after intranasal mucosal delivery of viral gene transfer vector. To address this issue, we have used fluorescence macroscopic imaging, luciferase quantification and PCR approaches to compare the relative distribution of transgene products or adenoviral gene sequences in the respiratory tract, lung, draining lymph nodes, olfactory bulb, brain and spleen. Intranasal mucosal delivery of replication-defective recombinant adenoviral vector results in gene transfer predominantly in the respiratory system including the lung while it does lead to a moderate level of gene transfer in the olfactory bulb. However, intranasal inoculation of adenoviral vector leads to little or no viral dissemination to the major region of the CNS, the brain. These experimental findings support the efficaciousness of intranasal adenoviral-mediated gene transfer for the purpose of mucosal immunization and suggest that it may not be of significant safety concern.

Differentially imprinted innate immunity by mucosal boost vaccination determines antituberculosis immune protective outcomes, independent of T-cell immunity

Homologous and heterologous parenteral prime–mucosal boost immunizations have shown great promise in combating mucosal infections such as tuberculosis and AIDS. However, their immune mechanisms remain poorly defined. In particular, it is still unclear whether T-cell and innate immunity may be independently affected by these immunization modalities and how it impacts immune protective outcome. Using two virus-based tuberculosis vaccines (adenovirus (Ad) and vesicular stomatitis virus (VSV) vectors), we found that while both homologous (Ad/Ad) and heterologous (Ad/VSV) respiratory mucosal boost immunizations elicited similar T-cell responses in the lung, they led to drastically different immune protective outcomes. Compared with Ad-based boosting, VSV-based boosting resulted in poorly enhanced protection against tuberculosis. Such inferior protection was associated with differentially imprinted innate phagocytes, particularly the CD11c+CD11b+/− cells, in the lung. We identified heightened type 1 interferon (IFN) responses to be the triggering mechanism. Thus, increased IFN-β severely blunted interleukin-12 responses in infected phagocytes, which in turn impaired their nitric oxide production and antimycobacterial activities. Our study reveals that vaccine vectors may differentially imprint innate cells at the mucosal site of immunization, which can impact immune-protective outcome, independent of T-cell immunity, and it is of importance to determine both T-cell and innate cell immunity in vaccine studies.

Pulmonary M. tuberculosis infection delays Th1 immunity via immunoadaptor DAP12-regulated IRAK-M and IL-10 expression in antigen- presenting cells

Interaction of mycobacteria with the host leads to retarded expression of T helper cell type 1 (Th1) immunity in the lung. However, the immune mechanisms remain poorly understood. Using in vivo and in vitro models of Mycobacterium tuberculosis (M. tb) infection, we find the immunoadaptor DAP12 (DNAX-activating protein of 12 kDa) in antigen-presenting cells (APCs) to be critically involved in this process. Upon infection of APCs, DAP12 is required for IRAK-M (interleukin-1 receptor-associated kinase M) expression, which in turn induces interleukin-10 (IL-10) and an immune-suppressed phenotype of APCs, thus leading to suppressed Th1 cell activation. Lack of DAP12 reduces APC IL-10 production and increases their Th1 cell-activating capability, resulting in expedited Th1 responses and enhanced protection. On the other hand, adoptively transferred DAP12-competent APCs suppress Th1 cell activation within DAP12-deficient hosts, and blockade of IL-10 aborts the ability of DAP12-competent APCs to suppress Th1 activation. Our study identifies the DAP12/IRAK-M/IL-10 to be a novel molecular pathway in APCs exploited by mycobacterial pathogens, allowing infection a foothold in the lung.