Patrick C. Reading

Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus

Effective immunity is dependent on long-surviving memory T cells. Various memory subsets make distinct contributions to immune protection, especially in peripheral infection. It has been suggested that T cells in nonlymphoid tissues are important during local infection, although their relationship with populations in the circulation remains poorly defined. Here we describe a unique memory T cell subset present after acute infection with herpes simplex virus that remained resident in the skin and in latently infected sensory ganglia. These T cells were in disequilibrium with the circulating lymphocyte pool and controlled new infection with this virus. Thus, these cells represent an example of tissue-resident memory T cells that can provide protective immunity at points of pathogen entry.

The Poxvirus Protein A52R Targets Toll-like Receptor Signaling Complexes to Suppress Host Defense

Toll-like receptors (TLRs) are crucial in the innate immune response to pathogens, in that they recognize and respond to pathogen associated molecular patterns, which leads to activation of intracellular signaling pathways and altered gene expression. Vaccinia virus (VV), the poxvirus used to vaccinate against smallpox, encodes proteins that antagonize important components of host antiviral defense. Here we show that the VV protein A52R blocks the activation of the transcription factor nuclear factor κB (NF-κB) by multiple TLRs, including TLR3, a recently identified receptor for viral RNA. A52R associates with both interleukin 1 receptor–associated kinase 2 (IRAK2) and tumor necrosis factor receptor–associated factor 6 (TRAF6), two key proteins important in TLR signal transduction. Further, A52R could disrupt signaling complexes containing these proteins. A virus deletion mutant lacking the A52R gene was attenuated compared with wild-type and revertant controls in a murine intranasal model of infection. This study reveals a novel mechanism used by VV to suppress the host immunity. We demonstrate viral disabling of TLRs, providing further evidence for an important role for this family of receptors in the antiviral response.

Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence

Viral immune evasion strategies target key aspects of the host antiviral response. Recently, it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic interactions between the Toll-like–interleukin-1 resistance (TIR) domains of the receptors and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain–containing protein identified to date. We demonstrate that A46R targets the host TIR adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain–containing adaptor inducing IFN-β (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor κB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-β by TLR3 and TLR4 and suppresses VV replication in macrophages. Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show that A46R is functionally distinct from another described VV TLR inhibitor, A52R. Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model, demonstrating the importance of A46R for VV virulence.

Neutrophils Ameliorate Lung Injury and the Development of Severe Disease during Influenza Infection

The clinical response to influenza infection ranges from mild disease to severe pneumonia and it remains unclear whether the inflammatory response to infection is protective or pathogenic. We have defined a novel role for neutrophils in ameliorating lung injury during influenza infection, thereby limiting development of severe disease. Infection of neutrophil-depleted mice with influenza virus HKx31 (H3N2) led to rapid weight loss, pneumonia, and death. Neutropenia was associated with enhanced virus replication in the respiratory tract; however, viral titers were declining at the time of death, leading us to investigate other factors contributing to mortality. In addition to thymic atrophy, lymphopenia, and viremic spread, depletion of neutrophils led to exacerbated pulmonary inflammation, edema, and respiratory dysfunction. Thus, while it is well established that neutrophils contribute to lung injury in a range of pathological conditions, reduced numbers or impaired neutrophil function can facilitate progression of mild influenza to severe clinical disease.

Involvement of the Mannose Receptor in Infection of Macrophages by Influenza Virus

ABSTRACT Influenza viruses A/PR/8/34 (PR8; H1N1), A/Aichi/68 X-31 (HKx31; H3N2), and A/Beijing/89 X-109 (BJx109; H3N2) show marked differences in their ability to infect murine macrophages, including resident alveolar and peritoneal macrophages as well as the macrophage-derived cell line J774. The hierarchy in infectivity of the viruses (PR8 < HKx31 < BJx109) resembles that of their reactivity with mannose-binding lectins of the collectin family. Since the macrophage mannose receptor recognizes the same spectrum of monosaccharides as the collectins do, we investigated the possible involvement of this receptor in infection of macrophages by influenza virus. In competitive binding studies, the binding of 125I-labeled mannosylated bovine serum albumin to macrophages was inhibited by the purified hemagglutinin and neuraminidase (HANA) glycoproteins of influenza virus but not by HANA that had been treated with periodate to oxidize its oligosaccharide side chains. The inhibitory activity of HANA from the three strains of virus differed markedly and correlated with the infectivity of each virus for macrophages. Infection of macrophages, but not MDCK cells, by influenza virus was inhibited by yeast mannan. A variant line of J774 cells, J774E, which expresses elevated levels of the mannose receptor, was more readily infected than J774, and the sensitivity of J774E cells to infection was greatly reduced by culture in the presence of d-mannose, which down-modulated mannose receptor expression. Together, the data implicate the mannose receptor as a major endocytic receptor in the infectious entry of influenza virus, and perhaps other enveloped viruses, into murine macrophages.

Cross-Reactive Influenza-Specific Antibody-Dependent Cellular Cytotoxicity Antibodies in the Absence of Neutralizing Antibodies

A better understanding of immunity to influenza virus is needed to generate cross-protective vaccines. Engagement of Ab-dependent cellular cytotoxicity (ADCC) Abs by NK cells leads to killing of virus-infected cells and secretion of antiviral cytokines and chemokines. ADCC Abs may target more conserved influenza virus Ags compared with neutralizing Abs. There has been minimal interest in influenza-specific ADCC in recent decades. In this study, we developed novel assays to assess the specificity and function of influenza-specific ADCC Abs. We found that healthy influenza-seropositive young adults without detectable neutralizing Abs to the hemagglutinin of the 1968 H3N2 influenza strain (A/Aichi/2/1968) almost always had ADCC Abs that triggered NK cell activation and in vitro elimination of influenza-infected human blood and respiratory epithelial cells. Furthermore, we detected ADCC in the absence of neutralization to both the recent H1N1 pandemic strain (A/California/04/2009) as well as the avian H5N1 influenza hemagglutinin (A/Anhui/01/2005). We conclude that there is a remarkable degree of cross-reactivity of influenza-specific ADCC Abs in seropositive humans. Targeting cross-reactive influenza-specific ADCC epitopes by vaccination could lead to improved influenza vaccines.

Critical Role of Airway Macrophages in Modulating Disease Severity during Influenza Virus Infection of Mice

ABSTRACT Airway macrophages provide a first line of host defense against a range of airborne pathogens, including influenza virus. In this study, we show that influenza viruses differ markedly in their abilities to infect murine macrophages in vitro and that infection of macrophages is nonproductive and no infectious virus is released. Virus strain BJx109 (H3N2) infected macrophages with high efficiency and was associated with mild disease following intranasal infection of mice. In contrast, virus strain PR8 (H1N1) was poor in its ability to infect macrophages and highly virulent for mice. Depletion of airway macrophages by clodronate-loaded liposomes led to the development of severe viral pneumonia in BJx109-infected mice but did not modulate disease severity in PR8-infected mice. The severe disease observed in macrophage-depleted mice infected with BJx109 was associated with exacerbated virus replication in the airways, leading to severe airway inflammation, pulmonary edema, and vascular leakage, indicative of lung injury. Thymic atrophy, lymphopenia, and dysregulated cytokine and chemokine production were additional systemic manifestations associated with severe disease. Thus, airway macrophages play a critical role in limiting lung injury and associated disease caused by BJx109. Furthermore, the inability of PR8 to infect airway macrophages may be a critical factor contributing to its virulence for mice.

Antiviral Activity of the Long Chain Pentraxin PTX3 against Influenza Viruses

Proteins of the innate immune system can act as natural inhibitors of influenza virus, limiting growth and spread of the virus in the early stages of infection before the induction of adaptive immune responses. In this study, we identify the long pentraxin PTX3 as a potent innate inhibitor of influenza viruses both in vitro and in vivo. Human and murine PTX3 bound to influenza virus and mediated a range of antiviral activities, including inhibition of hemagglutination, neutralization of virus infectivity and inhibition of viral neuraminidase. Antiviral activity was associated with binding of the viral hemagglutinin glycoprotein to sialylated ligands present on PTX3. Using a mouse model we found PTX3 to be rapidly induced following influenza infection and that PTX3−/− mice were more susceptible than wild-type mice to infection by PTX3-sensitive virus strains. Therapeutic treatment of mice with human PTX3 promoted survival and reduced viral load in the lungs following infection with PTX3-sensitive, but not PTX3-resistant, influenza viruses. Together, these studies describe a novel antiviral role for PTX3 in early host defense against influenza infections both in vitro and in vivo and describe the therapeutic potential of PTX3 in ameliorating disease during influenza infection.

Dermal infection with vaccinia virus reveals roles for virus proteins not seen using other inoculation routes

Previously, we developed a model for testing the virulence and immunogenicity of vaccinia virus (VV) mutants based on the intradermal injection of BALB/c mouse ear pinnae. The model is characterized by a local infection in the inoculated skin without signs of systemic illness, mimicking dermal vaccination with VV. Here a further characterization of this model is presented, including the responses of mice to infectious virus doses as low as 10 p.f.u., a quantification of the infiltrate at the site of infection and use of different virus and mouse strains. The model was then used to compare the pathogenesis of six mutants of VV strain Western Reserve (WR) lacking genes A36R, A40R, A44L, B12R, B13R or B15R with that of appropriate control viruses. All of these genes except B12R and B15R influence the outcome of dermal infection with WR and for A40R and B13R this is the first role that has been reported after infection of mammals. A comparison of new and published results from intradermal and intranasal models is presented, showing that out of 16 gene deletion or insertion mutants of VV, half have phenotypes distinct from controls in only one of these models. Thus, the intranasal and intradermal models are complementary tools for dissecting the genetic basis of VV virulence.

Complement-dependent neutralization of influenza virus by a serum mannose-binding lectin

The nature of the beta inhibitor in guinea-pig serum and its mechanism of neutralization of influenza virus have been investigated. This inhibitor was shown to be a mannose-binding lectin serologically related to human serum mannose-binding protein. Ca(2+)-dependent binding of the guinea-pig lectin to influenza virus or to mannan could be detected with polyclonal or monoclonal antibodies against human mannose-binding protein in an ELISA. Furthermore, the monoclonal antibody inhibited both the haemagglutination-inhibiting and virus-neutralizing activities of the guinea-pig lectin. The lectin was active against influenza viruses of both type A and type B. In haemagglutination inhibition it acts independently of complement, apparently by sterically hindering access to the receptor-binding site on the viral haemagglutinin through binding of the lectin to carbohydrate side-chains in the vicinity of this site. Neutralization by the lectin, however, was shown to require activation of the classical complement pathway. To our knowledge, the neutralization of influenza virus by a serum lectin plus complement represents a previously unrecognized mechanism of complement-dependent viral inactivation that may be important in first-line host defence against a variety of enveloped viruses.