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

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.Airway macrophages (Mφ) (AM), located at the interphase between air and lung tissue, provide the first line of defense following inhalation of airborne pathogens, including influenza viruses. In addition to phagocytosis of virions and virus-infected cells (16, 24), infection of AM represents an early event in recognition of the virus by the innate immune system. Following intranasal (i.n.) infection of mice, influenza virus replicates productively in type II epithelial cells lining the respiratory tract. Murine Mφ are also susceptible to influenza virus infection and viral proteins are produced, but replication is abortive and infectious progeny are not released (52, 69), although recent studies suggest limited release from mouse Mφ exposed to highly pathogenic H1N1 and H5N1 viruses (44). Following exposure to influenza virus, Mφ synthesize and release proinflammatory cytokines and alpha/beta interferon (26, 27, 45, 55), which further limit viral replication and spread within the respiratory tract. Inflammatory responses in the airways must be tightly regulated to ensure rapid virus clearance while avoiding excessive or chronic inflammation that may damage the delicate tissue-air interface.Liposome-encapsulated dichloromethylene diphosphonate (clodronate or CL2-MDP) is taken up by phagocytic Mφ and accumulates in the cytosol, resulting in Mφ death and depletion (66). Administration of clodronate liposomes (CL-LIP) has been widely used to selectively deplete airway Mφ in mouse models without affecting circulating monocytes (for examples, see references 4, 6, 8, 36, 47, 64, and 71). While CL-LIP has been used predominantly in rodent models of infection, it is noteworthy that CL-LIP treatment of pigs, a natural host of influenza virus, resulted in enhanced morbidity and mortality following infection with a human H1N1 subtype virus (30). In murine studies, depletion of airway Mφ prior to influenza virus infection led to increased cytotoxic CD8⁺ T-cell responses in the lungs of virus-infected mice (71); however, treatment with CL-LIP 48 h after infection was associated with impaired CD8⁺ T-cell responses (41). Furthermore, CL-LIP treatment prior to intranasal infection of mice with a recombinant virus bearing the surface glycoproteins of the 1918 pandemic strain led to exacerbated disease and mortality (64). Treatment of mice with CL-LIP has been associated with enhanced virus replication (41, 64, 71); however, the mechanisms by which airway Mφ initiate and modulate inflammatory responses and disease after influenza virus infection have not been fully elucidated.We observed in a previous study that influenza A virus strains show marked differences in their abilities to infect murine Mφ in vitro and implicated the Mφ mannose receptor (MMR) (CD206), a C-type lectin (46), in infectious virus entry (48). Virus strain BJx109, a reassortant virus bearing the surface glycoproteins of A/Beijing/353/89 (H3N2) and internal components derived from A/PR/8/34 (H1N1) (PR8) bears a highly glycosylated hemagglutinin (HA) molecule and was shown to infect Mφ to high levels, while the HA of PR8 is poorly glycosylated and the virus infected Mφ very poorly. In the current study, we demonstrate that intranasal infection of mice with BJx109 leads to mild clinical disease, while infection with an equivalent dose of PR8 leads to severe disease and rapid death. Depletion of airway Mφ by intranasal administration of CL-LIP prior to and during infection with influenza viruses had little effect on the course of PR8 infection; however, BJx109 infection of Mφ-depleted animals led to severe disease and death. Severe disease was associated with enhanced virus replication, severe airway inflammation, and pulmonary edema and vascular leakage, indicative of lung injury. Together, these data demonstrate that airway Mφ play a critical role in moderating disease severity during BJx109 infection. Furthermore, they suggest that the ability of PR8 to evade infectious uptake by airway Mφ is likely to be an important factor contributing to its virulence for mice.     

Gamma Interferon Signaling in Macrophage Lineage Cells Regulates Central Nervous System Inflammation and Chemokine Production

Intracranial (i.c.) infection of mice with lymphocytic choriomeningitis virus (LCMV) results in anorexic weight loss, mediated by T cells and gamma interferon (IFN-γ). Here, we assessed the role of CD4⁺ T cells and IFN-γ on immune cell recruitment and proinflammatory cytokine/chemokine production in the central nervous system (CNS) after i.c. LCMV infection. We found that T-cell-depleted mice had decreased recruitment of hematopoietic cells to the CNS and diminished levels of IFN-γ, CCL2 (MCP-1), CCL3 (MIP-1α), and CCL5 (RANTES) in the cerebrospinal fluid (CSF). Mice deficient in IFN-γ had decreased CSF levels of CCL3, CCL5, and CXCL10 (IP-10), and decreased activation of both resident CNS and infiltrating antigen-presenting cells (APCs). The effects of IFN-γ signaling on macrophage lineage cells was assessed using transgenic mice, called “macrophages insensitive to interferon gamma” (MIIG) mice, that express a dominant-negative IFN-γ receptor under the control of the CD68 promoter. MIIG mice had decreased levels of CCL2, CCL3, CCL5, and CXCL10 compared to controls despite having normal numbers of LCMV-specific CD4⁺ T cells in the CNS. MIIG mice also had decreased recruitment of infiltrating macrophages and decreased activation of both resident CNS and infiltrating APCs. Finally, MIIG mice were significantly protected from LCMV-induced anorexia and weight loss. Thus, these data suggest that CD4⁺ T-cell production of IFN-γ promotes signaling in macrophage lineage cells, which control (i) the production of proinflammatory cytokines and chemokines, (ii) the recruitment of macrophages to the CNS, (iii) the activation of resident CNS and infiltrating APC populations, and (iv) anorexic weight loss.Immune cell recruitment to and infiltration of the central nervous system (CNS) is central to the pathology of a variety of inflammatory neurological diseases, including infectious meningoencephalitis, multiple sclerosis, and cerebral ischemia (59, 60). Chemokines have been shown to be highly upregulated in both human diseases and animal models of neuroinflammation and are thought to be important mediators of immune cell entry into the CNS (59, 60). For example, during experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS), the chemokines CCL2 (monocyte chemoattractant protein 1 [MCP-1α]), CCL3 (macrophage inflammatory protein 1α [MIP-1α]), CCL5 (regulated upon activation, T-cell expressed and secreted [RANTES]), and CXCL10 (gamma interferon [IFN-γ]-inducible protein 10 [IP-10]) are produced by either resident CNS cells or infiltrating cells (27) and serve to amplify the ongoing inflammatory response (25, 28). However, in some EAE studies, neither CCL3 nor CXCL10 were required for disease (72, 73). During CNS viral infection, CXCL10 and CCL5 are highly produced in several models (2, 41, 48, 82). In addition, mice deficient in CCR5, which binds (among others) CCL3 and CCL5, do not display impaired CNS inflammation after certain viral infections (13). Thus, the role of chemokines in CNS inflammation is likely complex and dissimilar between autoimmune and viral infection models.IFN-γ is present in the CNS during autoimmunity and infection (7, 54, 69). Several studies suggest that IFN-γ can be a potent inducer of CNS chemokine expression. Adenoviral expression of IFN-γ in the CNS strongly induced CCL5 and CXCL10 mRNA and protein, and this induction was dependent on the presence of the IFN-γ receptor (50). In EAE and Toxoplasma infection, mice deficient in IFN-γ or the IFN-γ receptor demonstrated reduced expression of several chemokines, including CCL2, CCL3, CCL5, and CXCL10 (26, 69). However, given the near-ubiquitous expression of the IFN-γ receptor (44), the mechanisms by which IFN-γ regulates CNS chemokine production remain to be elucidated.We studied neuroinflammation and immune-mediated disease using a well-studied mouse model of infection with lymphocytic choriomeningitis virus (LCMV). Intracranial (i.c.) injection of mice with LCMV results in seizures and death 6 to 8 days after inoculation. The onset of symptoms is associated with a massive influx of mononuclear cells into the cerebrospinal fluid (CSF), meninges, choroid plexus, and ependymal membranes (6, 8, 18), as well as the presence of proinflammatory cytokines (7, 38). The immune response is critical for disease, since infection of irradiated or T-cell-depleted mice leads to persistent infection with very high levels of virus in multiple tissues without the development of lethal meningitis (18, 34, 64). i.c. LCMV infection of β₂-microglobulin-deficient mice (β₂m^−/− mice) also results in meningitis and production of proinflammatory cytokines and chemokines; however, meningitis occurs with a later onset and lower severity compared to wild-type mice (17, 24, 53, 57). Interestingly, i.c. LCMV infection of these mice also causes severe anorexia and weight loss (33, 38, 46, 52, 57) that is mediated by major histocompatibility complex (MHC) class II-restricted, CD4⁺ T cells (17, 46, 53, 57). Anorexia and weight loss are also observed in wild-type mice, but they succumb to lethal meningitis shortly thereafter (33), making study of this particular aspect of disease difficult. LCMV-induced weight loss, similar to what we have observed in β₂m^−/− mice also occurs in perforin-deficient mice, which possess CD8⁺ T cells (37). Although some reports have observed weight loss after peripheral LCMV infection (11, 45), we note that these studies used high doses of the clone 13 strain of LCMV, in contrast to our studies which have used the Armstrong strain of LCMV and orders of magnitude less virus (33, 38, 46, 52, 57). Although we cannot exclude a contribution of peripheral cells to weight loss in our i.c. Armstrong infection model, we previously showed that this weight loss does not occur with peripheral infection with LCMV Armstrong (33, 38), indicating that interactions between the CNS and the immune system are contribute substantially to disease.During LCMV infection, there is biphasic production of IFN-γ: a small, early peak of IFN-γ (most likely produced by NK or NKT cells), followed by T-cell-mediated production of IFN-γ (23, 75). Further, both CD4⁺ T cells and CD8⁺ T cells produce large amounts of IFN-γ after LCMV infection and T-cell production of IFN-γ is critical for LCMV-induced weight loss (35). Chemokines, especially CXCL10, CCL5, and CCL2, and their receptors, are upregulated in the brain after i.c. LCMV infection (2, 13). Brain chemokine mRNA expression after i.c. LCMV infection is reduced in IFN-γ-deficient mice and relatively absent in athymic mice (2). However, the mechanism(s) by which T cells and IFN-γ mediate the effects on CNS chemokine expression, cellular infiltration into the CNS, and LCMV-induced anorexic weight loss remain unclear.In the present study, we focused on two major questions. The first question concerned the role of IFN-γ on immune cell recruitment to and chemokine/cytokine production within the CNS? We found that macrophages and myeloid dendritic cells (DCs) require IFN-γ for their accumulation within the CNS. Second, since macrophages and myeloid DCs are the predominant cellular infiltrate, we sought to determine whether IFN-γ signaling on these cells was direct with regard to their recruitment and to chemokine/cytokine production. We found that IFN-γ signaling in macrophage lineage cells contributes significantly to their recruitment, to chemokine production in the CNS, and to anorexic weight loss. Together, these data suggest that much of the proinflammatory effects of IFN-γ in the CNS are mediated by the effects of IFN-γ on CD68-bearing cells.     

Receptor Specificity of Influenza A H3N2 Viruses Isolated in Mammalian Cells and Embryonated Chicken Eggs

Isolation of human subtype H3N2 influenza viruses in embryonated chicken eggs yields viruses with amino acid substitutions in the hemagglutinin (HA) that often affect binding to sialic acid receptors. We used a glycan array approach to analyze the repertoire of sialylated glycans recognized by viruses from the same clinical specimen isolated in eggs or cell cultures. The binding profiles of whole virions to 85 sialoglycans on the microarray allowed the categorization of cell isolates into two groups. Group 1 cell isolates displayed binding to a restricted set of α2-6 and α2-3 sialoglycans, whereas group 2 cell isolates revealed receptor specificity broader than that of their egg counterparts. Egg isolates from group 1 showed binding specificities similar to those of cell isolates, whereas group 2 egg isolates showed a significantly reduced binding to α2-6- and α2-3-type receptors but retained substantial binding to specific O- and N-linked α2-3 glycans, including α2-3GalNAc and fucosylated α2-3 glycans (including sialyl Lewis x), both of which may be important receptors for H3N2 virus replication in eggs. These results revealed an unexpected diversity in receptor binding specificities among recent H3N2 viruses, with distinct patterns of amino acid substitution in the HA occurring upon isolation and/or propagation in eggs. These findings also suggest that clinical specimens containing viruses with group 1-like receptor binding profiles would be less prone to undergoing receptor binding or antigenic changes upon isolation in eggs. Screening cell isolates for appropriate receptor binding properties might help focus efforts to isolate the most suitable viruses in eggs for production of antigenically well-matched influenza vaccines.Influenza A viruses are generally isolated and propagated in embryonated chicken eggs or in cultures of cells of mammalian origin. Human influenza viruses were previously noted to acquire mutations in the hemagglutinin (HA) gene upon isolation and culture in the allantoic sac of embryonated chicken eggs (herein simply referred to as “eggs”) compared to the sequences of those isolated in mammalian cell substrates (herein referred to as “cells”) (29, 30, 44, 53, 58). These mutations resulted in amino acid substitutions that were found to mediate receptor specificity changes and improved viral replication efficiency in eggs (37). In general, cell-grown viruses are assumed to be more similar than their egg-grown counterparts to the viruses present in respiratory secretions (30, 56). Since their emergence in 1968, influenza A (H3N2) viruses have evolved and adapted to the human host while losing their ability to be efficiently isolated and replicate in eggs, particularly after 1992 (37, 42, 48). The rate of isolation of H3N2 clinical specimens after inoculation into eggs can be up to ∼30 times lower than that in mammalian cell cultures, highlighting the strong selective pressure for the emergence of sequence variants (77).Virtually all influenza vaccines for human use were licensed decades ago by national regulatory authorities, which used a product manufactured from influenza viruses isolated and propagated exclusively in eggs; therefore, cell culture isolates have been unacceptable for this purpose (41, 71). The antigen composition of influenza vaccines requires frequent updates (every 2 years, on average) to closely match their antigenic properties to the most prevalent circulating antigenic drift variant viruses (51). The limited availability of H3N2 viruses isolated in eggs has on one or more occasions delayed vaccine composition updates and may have reduced the efficacy of vaccination against new antigenically drifted viruses (3, 34, 37).Entry of influenza viruses into host cells is mediated by HA, which binds to sialic acid containing glycoconjugates on the surface of epithelial cells in the upper respiratory tract (2, 13). The nature of the linkage between sialic acid and the vicinal sugar (usually galactose) varies in different host species and tissues and may therefore determine whether an influenza virus binds to and infects avian or human cells (40, 46, 59, 62, 72-75). Human influenza viruses preferentially bind to α2-6-linked sialic acids, and avian viruses predominantly bind to α2-3-linked sialic acids (59). Previous studies with chicken embryo chorioallantoic membranes revealed differential lectin binding, suggesting that α2-3-linked but not α2-6-linked sialosides are present on the epithelial cells (28). Human H3N2 viruses isolated in cell culture were reported to bind with a high affinity to α2-6-linked sialosides, while viruses isolated in eggs often had increased specificity for α2-3-linked sialosides (19, 20, 28). The functional classification of avian and mammalian influenza virus receptors is further complicated since in vitro and tissue-binding assays have led to new working hypotheses involving glycan chain length, topology, and the composition of the inner fragments of the carbohydrate chain as additional receptor specificity determinants (9, 17, 65, 66, 82). However, the significance of these in vitro properties remains unknown, since the structures of the natural sialosides on host cells that are used for infectious virus entry are undefined.The techniques most widely used to study the interactions of the influenza virus with host cell receptors employ animal cells in various assay formats (36, 57, 59, 64, 69). To overcome the problems of cell-based techniques, new assays that rely on labeled sialyl-glycoproteins or polymeric sialoglycans have been developed (18). However, these assays are limited by having only a few glycans available in polymeric form and offer low throughput. In contrast, glycan microarrays can assess virus binding to multiple well-defined glycans simultaneously. Previous work with influenza live or β-propiolactone (BPL)-inactivated virions as well as recombinantly produced HAs revealed a good correlation with receptor specificity compared to that achieved by other methods of analysis (4, 11, 57, 58, 65-68).Here we have compared paired isolates derived in eggs or cell cultures from the single clinical specimen to better understand their receptor binding specificity and its implications for vaccine production. We examined the differences in the sequences of the HAs between egg- and cell-grown isolates and analyzed their receptor binding profiles using glycan microarrays. Sequence analysis of the HA and glycan binding results revealed two distinct groups of viruses, with many egg isolates showing unexpectedly reduced levels of binding to α2-3 and α2-6 sialosides compared to the levels for the viruses isolated in mammalian cells. Furthermore, these studies highlighted that specific glycans may be important for H3N2 virus growth in eggs.     

Dynamics of Influenza Virus Infection and Pathology

A key question in pandemic influenza is the relative roles of innate immunity and target cell depletion in limiting primary infection and modulating pathology. Here, we model these interactions using detailed data from equine influenza virus infection, combining viral and immune (type I interferon) kinetics with estimates of cell depletion. The resulting dynamics indicate a powerful role for innate immunity in controlling the rapid peak in virus shedding. As a corollary, cells are much less depleted than suggested by a model of human influenza based only on virus-shedding data. We then explore how differences in the influence of viral proteins on interferon kinetics can account for the observed spectrum of virus shedding, immune response, and influenza pathology. In particular, induction of high levels of interferon (“cytokine storms”), coupled with evasion of its effects, could lead to severe pathology, as hypothesized for some fatal cases of influenza.Influenza A virus causes an acute respiratory disease in humans and other mammals; in humans, it is particularly important because of the rapidity with which epidemics develop, its widespread morbidity, and the seriousness of complications. Every year, an estimated 500,000 deaths worldwide, primarily of young children and the elderly, are attributed to seasonal influenza virus infections (49). Influenza pandemics may occur when an influenza virus with new surface proteins emerges, against which the majority of the population has no preexisting immunity. Both the emergence of H5N1 virus (34) and the current H1N1 virus pandemic (43) underline the importance of understanding the dynamics of infection and disease. A key question is, what regulates virus abundance in an individual host, causing the characteristic rapid decline in virus shedding following its initial peak? The main contenders in primary influenza virus infection are depletion of susceptible target cells and the impact of the host''s innate immune response (2, 20).On infection, the influenza virus elicits an immune response, including a rapid innate response that is correlated with the observed decline in the virus load after the first 2 days of infection (1). The slower adaptive response, including both humoral and cell-mediated components, takes several days to consolidate but is important for complete virus clearance and establishment of protective immunity. During infection of an immunologically naïve host, the innate immune response is particularly important as the first line of defense against infection. The innate immune response is regulated by chemokines and cytokines, chemical messengers produced by virus-infected epithelial cells and leukocytes (23), and natural interferon-producing cells, such as plasmacytoid dendritic cells (13). Among the key cytokines induced by epithelial cells infected with influenza A virus are type I interferons (IFNs) (IFN-α/β) (23), which directly contribute to the antiviral effect on infected and neighboring cells (38).Like other viruses, influenza A viruses have evolved strategies to limit the induction of innate immune responses (38). The NS1 protein plays a dominant role, and without it, the virus is unable to grow well or to cause pathology in an immunocompetent host (14). NS1 is multifunctional and counteracts both the induction of IFN expression and the function of IFN-activated antiviral effectors via multiple mechanisms (12, 17). Individual strains of influenza A virus possess these activities to various degrees (15, 21, 22, 26), and accordingly, NS1 has been implicated as a virulence factor (3, 17). A striking effect of the failure to control the innate response to virus infection is seen as a “cytokine storm,” which causes severe pathology (8).While there is an extensive literature on modeling influenza virus spread at the population level, the individual-host scale has received much less attention (2, 4, 5, 18, 19, 20, 27, 28). In a recent important paper, Baccam et al. modeled the kinetics of influenza A virus (2). The innate dynamics were included in the form of an IFN response that delayed and reduced virus production but did not prevent it; thus, the infection was resolved primarily through near-total depletion of epithelial cells. Their model was fitted to virus titers from human volunteers exposed to H1N1 influenza virus, but no data were available on the innate immune response or epithelial cell pathology. This has been a general difficulty in developing and validating more refined within-host models; there is a lack of detailed biological data from natural host systems, in particular, measures of immune kinetics and patterns of cellular depletion.The model presented here explicitly includes the ability of IFN to induce a fully antiviral state in order to explore the relative regulatory role of innate immunity and target cell depletion. Data from experimental infections of immunologically naïve horses with an equine influenza virus (36) allowed us to calibrate our model, not only to viral kinetics, but also to IFN dynamics and cell depletion in the context of infection of a naïve natural mammalian host. With our fitted model, we then investigate modulation of the immune response.     

Interleukin-15 Is Critical in the Pathogenesis of Influenza A Virus-Induced Acute Lung Injury

Highly pathogenic influenza A viruses cause acute severe pneumonia to which the occurrence of “cytokine storm” has been proposed to contribute. Here we show that interleukin-15 (IL-15) knockout (KO) mice exhibited reduced mortality after infection with influenza virus A/FM/1/47 (H1N1, a mouse-adapted strain) albeit the viral titers of these mice showed no difference from those of control mice. There were significantly fewer antigen-specific CD44⁺ CD8⁺ T cells in the lungs of infected IL-15 KO mice, and adoptive transfer of the CD8⁺ T cells caused reduced survival of IL-15 KO mice following influenza virus infection. Mice deficient in β₂-microglobulin by gene targeting and those depleted of CD8⁺ T cells by in vivo administration of anti-CD8 monoclonal antibody displayed a reduced mortality rate after infection. These results indicate that IL-15-dependent CD8⁺ T cells are at least partly responsible for the pathogenesis of acute pneumonia caused by influenza A virus.Highly pathogenic influenza A viruses cause acute severe pneumonia that results in high morbidity and significant mortality (11, 12, 24, 26). Elevated levels of serum cytokines and chemokines accompany these clinical manifestations, and the possibility that this “cytokine storm” contributes to increased severity of the disease caused by avian H5N1 virus and by other strains of influenza A virus has been proposed (10, 21, 33). In fact, CCR2-deficient mice [CCR2 is chemokine (C-C motif) receptor 2] were protected from early pathological manifestations despite higher pulmonary titers of the influenza virus A/PR/8/34 (H1N1) strain (7). Tumor necrosis factor receptor 1 (TNFR-1)-deficient mice exhibited significantly reduced morbidity following challenge with H5N1 virus (31). Other cytokines or chemokines have also been investigated (8, 28, 34, 35, 38). Thus, at least some of the elevated proinflammatory cytokines may contribute to the pathogenesis of influenza A virus.Interleukin-15 (IL-15) is a pleiotropic cytokine involved in both innate and adaptive immune responses (20, 36). IL-15 utilizes the β-chain of the IL-2 receptor (IL-2R) (CD122) and the common cytokine receptor γ-chain (CD132) for signal transduction in lymphocytes and therefore shares many biological properties with IL-2 (3). Memory CD8⁺ T cells, natural killer (NK) cells, NKT cells, and intraepithelial lymphocyte (IEL) T cells (15, 23, 42) decrease in mice with defective IL-15 signaling, indicating the importance of IL-15 in their development and/or maintenance. IL-15 regulates not only the number of memory CD8⁺ T cells but also activation of their functions, including gamma interferon (IFN-γ) production and cytotoxic activity (40), which are important to target the virus (9). Therefore, it is possible that we may be able to use IL-15 as an immune-enhancing molecular adjuvant in vaccines for protection against various pathogens, including influenza A virus (37).In the present study, we demonstrate that IL-15 knockout (KO) mice exhibited high resistance against infection with mouse-adapted influenza virus A/FM/1/47 (H1N1) strain. We show for the first time that IL-15-dependent CD8⁺ T cells are at least partly responsible for the pathogenesis of acute pneumonia caused by influenza A virus. In addition, our observations are important in the light of recent research into the use of IL-15 as an adjuvant for vaccination.     

Pandemic H1N1 2009 Influenza A Virus Induces Weak Cytokine Responses in Human Macrophages and Dendritic Cells and Is Highly Sensitive to the Antiviral Actions of Interferons

In less than 3 months after the first cases of swine origin 2009 influenza A (H1N1) virus infections were reported from Mexico, WHO declared a pandemic. The pandemic virus is antigenically distinct from seasonal influenza viruses, and the majority of human population lacks immunity against this virus. We have studied the activation of innate immune responses in pandemic virus-infected human monocyte-derived dendritic cells (DC) and macrophages. Pandemic A/Finland/553/2009 virus, representing a typical North American/European lineage virus, replicated very well in these cells. The pandemic virus, as well as the seasonal A/Brisbane/59/07 (H1N1) and A/New Caledonia/20/99 (H1N1) viruses, induced type I (alpha/beta interferon [IFN-α/β]) and type III (IFN-λ1 to -λ3) IFN, CXCL10, and tumor necrosis factor alpha (TNF-α) gene expression weakly in DCs. Mouse-adapted A/WSN/33 (H1N1) and human A/Udorn/72 (H3N2) viruses, instead, induced efficiently the expression of antiviral and proinflammatory genes. Both IFN-α and IFN-β inhibited the replication of the pandemic (H1N1) virus. The potential of IFN-λ3 to inhibit viral replication was lower than that of type I IFNs. However, the pandemic virus was more sensitive to the antiviral IFN-λ3 than the seasonal A/Brisbane/59/07 (H1N1) virus. The present study demonstrates that the novel pandemic (H1N1) influenza A virus can readily replicate in human primary DCs and macrophages and efficiently avoid the activation of innate antiviral responses. It is, however, highly sensitive to the antiviral actions of IFNs, which may provide us an additional means to treat severe cases of infection especially if significant drug resistance emerges.The novel swine origin 2009 influenza A (H1N1) virus was identified in April 2009, and it is currently causing the first influenza pandemic of the 21st century. The virus is a completely new reassortant virus (8, 38), and the majority of the human population does not have preexisting immunity against it. The case fatality rate of the current pandemic virus infection is still unclear, but it is estimated to be somewhat higher than that of seasonal influenza virus infections (8). In most cases, the pandemic 2009 A (H1N1) virus causes an uncomplicated respiratory tract illness with symptoms similar to those caused by seasonal influenza viruses. However, gastrointestinal symptoms atypical to seasonal influenza have been detected in a significant proportion of cases (4, 7, 35).The pandemic 2009 (H1N1) influenza A virus originates from a swine influenza A virus strain. It underwent multiple reassortment events in pigs and then transferred into the human population (8, 38). The new virus has gene segments from the North American triple-reassortant and Eurasian swine H1N1 viruses (8, 38). Sequence analysis of this new pandemic virus revealed that hemagglutinin (HA), NP, and NS gene segments are derived from the classical swine viruses, PB1 from human H3N2, and PB2 and PA from avian viruses within the triple-reassortant virus (8). In addition, the NA and M segments originate from the Eurasian swine virus lineage. The pandemic 2009 (H1N1) virus is genetically and antigenically distinct from previous seasonal human influenza A (H1N1) viruses. Thus, the current seasonal influenza vaccines are likely to give little, if any, protection against pandemic 2009 A (H1N1) virus infection (12, 14). However, some evidence indicates that people born early in the 20th century have cross-neutralizing antibodies against the pandemic 2009 A (H1N1) viruses (12, 14).At present, relatively little is known about the pathogenesis and transmission of the pandemic 2009 A (H1N1) virus in humans. Studies with ferrets revealed that the pandemic virus replicated better than seasonal H1N1 viruses in the respiratory tracts of the animals. This suggests that the virus is more pathogenic in ferrets than seasonal influenza viruses (19, 24). The respiratory tract is the primary infection site of all mammalian influenza viruses, and, indeed, the specific glycan receptors on the apical surface of the upper respiratory tract have been reported to bind HA of the 2009 A (H1N1) virus (19). In human lung tissue binding assays, 2009 A (H1N1) HA showed a glycan binding pattern similar to that of the HA from the pandemic 1918 A (H1N1) virus though its affinity to α2,6 glycans was much lower than that of the 1918 virus HA. The lower glycan binding properties of the pandemic 2009 A (H1N1) virus seemed to correlate with less-efficient transmission in ferrets compared to seasonal H1N1 viruses (19). According to another study with ferrets, the transmission of the pandemic 2009 A (H1N1) virus via respiratory droplets was as efficient as that of a seasonal A (H1N1) virus (24). It is clear that, besides experimental infections in animal models, analyses of the characters and pathogenesis of the pandemic 2009 A (H1N1) virus infection in humans are urgently needed.In the present study, we have focused on analyzing innate immune responses in primary human dendritic cells (DCs) and macrophages in response to an infection with one of the Finnish isolates of the pandemic 2009 A (H1N1) virus. DCs and macrophages reside beneath the epithelium of the respiratory organs, and these cells are thus potential targets for influenza viruses. From the epithelial cells influenza viruses spread in DCs and macrophages, which coordinate the development of an effective innate immune response against the virus (22, 34, 41). During influenza virus infection, DCs and macrophages secrete antiviral cytokines such as interferons (IFNs) and tumor necrosis factor alpha (TNF-α) (3, 13, 26). Moreover, DCs and macrophages activate virus-destroying NK cells and T cells with the cytokines they secrete and via direct cell-to-cell contacts (9, 29, 33, 37). Here we show that the pandemic (H1N1) virus infects and replicates very well in human monocyte-derived DCs and macrophages. The pandemic virus as well as two recent seasonal H1N1 viruses induced a relatively weak innate immune response in these cells, as evidenced by a poor expression of antiviral and proinflammatory cytokine genes. However, like seasonal influenza A viruses, the pandemic 2009 (H1N1) virus was extremely sensitive to the antiviral actions of type I IFNs (IFN-α/β). Interestingly, the pandemic 2009 (H1N1) virus was even more sensitive to antiviral IFN-λ3 than a seasonal A (H1N1) virus. Thus, IFNs may provide us with an additional means to combat severe pandemic influenza virus infections, especially if viral resistance against neuraminidase (NA) inhibitors begins to emerge.     

Interferon-Induced ISG15 Conjugation Inhibits Influenza A Virus Gene Expression and Replication in Human Cells

The ubiquitin-like ISG15 protein, as well as its conjugating enzymes, is induced by type I interferons (IFNs). Experiments using ISG15 knockout (ISG15^−/−) mice established that ISG15 and/or its conjugation inhibits the replication of influenza A virus. However, in contrast to the virus inhibition results for mice, the rates of virus replication in ISG15^+/+ and ISG15^−/− mouse embryo fibroblasts in tissue culture were similar. Here we focus on human tissue culture cells and on the effect of ISG15 and/or its conjugation on influenza A virus gene expression and replication in such cells. We demonstrate that IFN-induced antiviral activity against influenza A virus in human cells is significantly alleviated by inhibiting ISG15 conjugation using small interfering RNAs directed against ISG15-conjugating enzymes. IFN-induced antiviral activity against influenza A virus protein synthesis was reduced 5- to 20-fold by suppressing ISG15 conjugation. The amounts of the viral proteins that were restored by these siRNA treatments were approximately 40 to 50% of the amounts produced in cells that were not pretreated with IFN. Further, we show that ISG15 conjugation inhibits influenza A virus replication 10- to 20-fold at early times after infection in human cells. These results show that ISG15 conjugation plays a substantial role in the antiviral state induced by IFN in human cells. In contrast, we show that in mouse embryo fibroblasts ISG15 conjugation not only does not affect influenza A virus replication but also does not contribute to the IFN-induced antiviral activity against influenza A virus gene expression.Virus infection activates the synthesis of type I interferons (IFN-α and IFN-β), which induce the synthesis of a large array of proteins, many of which play crucial roles in the antiviral response (1). One of the most strongly induced proteins is ISG15, a 15-kDa ubiquitin-like protein that becomes conjugated to many cellular proteins (6, 8, 9, 12, 18, 22, 26, 30). Three of the human enzymes that catalyze this conjugation, the UbE1L E1 enzyme, the UbcH8 E2 enzyme, and the Herc5 E3 enzyme, are also induced by IFN-β (4, 10, 26, 27, 29). Although it had been reported that UbcH8 functions in both ISG15 and ubiquitin conjugation (3, 10, 13, 25, 28, 29), a recent study demonstrated that UbcH8 is unlikely to function in ubiquitin conjugation in vivo for two reasons: K_m measurements revealed that the E1 ubiquitin-activating enzyme, unlike UbE1L, exhibits very low affinity for UbcH8, and UbcH8 is poorly, if not at all, expressed in the absence of IFN treatment, indicating that UbcH8 functions only during the IFN response (5). A large number of human proteins that are targets for ISG15 conjugation have been identified (22, 26, 30). Most of these targets are constitutively expressed proteins that function in diverse cellular pathways, but several of the targets are IFN-α/-β-induced antiviral proteins.Because the NS1 protein of influenza B virus (NS1B) was shown to bind ISG15 and inhibit its conjugation to target proteins, it was proposed that ISG15 and/or its conjugation is inhibitory to the replication of influenza B virus (27). Subsequently, experiments using ISG15 knockout (ISG15^−/−) mice established that ISG15 and/or its conjugation inhibits the replication of not only influenza B virus but also influenza A virus (16). For example, at one of the inoculum levels employed for influenza A virus, 52% of the ISG15^−/− mice died, whereas a significantly smaller percentage, 23%, of the ISG15^+/+ mice died. However, the effect of ISG15 and/or its conjugation on influenza A virus replication was not detected in mouse embryo fibroblasts (MEFs) in tissue culture. MEFs supported only very limited replication of influenza A virus, and there was no significant difference in virus replication between ISG15^+/+ and ISG15^−/− MEFs (16). These investigators postulated that influenza A virus replication was probably selectively spared in other cell types of the ISG15^−/− mouse. A subsequent study showed that ISG15 conjugation exerts its antiviral action against influenza B virus (and presumably against influenza A virus) in radioresistant stromal cells of the mouse (14). However, an antiviral effect of ISG15 conjugation against influenza A virus has not yet been demonstrated in mouse cells in tissue culture.In the present study we focus on human tissue culture cells and on the effect of ISG15 and/or its conjugation on the replication of influenza A virus in such cells. We show that IFN-induced antiviral activity against influenza A virus in human cells is significantly alleviated by inhibiting ISG15 conjugation using small interfering RNAs (siRNAs) against ISG15-conjugating enzymes. Our results show that both the synthesis of viral proteins and the early rate of virus replication are inhibited by ISG15 conjugation. In contrast, we show that in MEFs ISG15 conjugation not only does not affect influenza A virus replication but also does not contribute to IFN-induced antiviral activity against influenza A virus gene expression.     

Ocular Infection of Mice with Influenza A (H7) Viruses: a Site of Primary Replication and Spread to the Respiratory Tract

Avian H7 influenza viruses have been responsible for poultry outbreaks worldwide and have resulted in numerous cases of human infection in recent years. The high rate of conjunctivitis associated with avian H7 subtype virus infections may represent a portal of entry for avian influenza viruses and highlights the need to better understand the apparent ocular tropism observed in humans. To study this, mice were inoculated by the ocular route with viruses of multiple subtypes and degrees of virulence. We found that in contrast to human (H3N2 and H1N1) viruses, H7N7 viruses isolated from The Netherlands in 2003 and H7N3 viruses isolated from British Columbia, Canada, in 2004, two subtypes that were highly virulent for poultry, replicated to a significant titer in the mouse eye. Remarkably, an H7N7 virus, as well as some avian H5N1 viruses, spread systemically following ocular inoculation, including to the brain, resulting in morbidity and mortality of mice. This correlated with efficient replication of highly pathogenic H7 and H5 subtypes in murine corneal epithelial sheets (ex vivo) and primary human corneal epithelial cells (in vitro). Influenza viruses were labeled to identify the virus attachment site in the mouse cornea. Although we found abundant H7 virus attachment to corneal epithelial tissue, this did not account for the differences in virus replication as multiple subtypes were able to attach to these cells. These findings demonstrate that avian influenza viruses within H7 and H5 subtypes are capable of using the eye as a portal of entry.Highly pathogenic avian influenza (HPAI) H5N1 viruses, which have resulted in over 420 documented cases of human infection to date, have generally caused acute, often severe and fatal, respiratory illness (1, 50). While conjunctivitis following infection with H5N1 or human influenza viruses has been rare, most human infections associated with H7 subtype viruses have resulted in ocular and not respiratory disease (1, 9, 37, 38). Infrequent reports of human conjunctivitis infection following exposure to H7 influenza viruses date from 1977, predominantly resulting from laboratory or occupational exposure (21, 40, 48). However, in The Netherlands in 2003, more than 80 human infections with H7N7 influenza virus occurred among poultry farmers and cullers amid widespread outbreaks of HPAI in domestic poultry; the majority of these human infections resulted in conjunctivitis (14, 20). Additionally, conjunctivitis was documented in the two human infections resulting from an H7N3 outbreak in British Columbia, Canada, in 2004, as well as in H7N3- and H7N2-infected individuals in the United Kingdom in 2006 and 2007, respectively (13, 18, 29, 46, 51). The properties that contribute to an apparent ocular tropism of some influenza viruses are currently not well understood (30).Host cell glycoproteins bearing sialic acids (SAs) are the cellular receptors for influenza viruses and can be found on epithelial cells within both the human respiratory tract and ocular tissue (26, 31, 41). Both respiratory and ocular tissues additionally secrete sialylated mucins that function in pathogen defense and protection of the epithelial surface (5, 11, 22). Within the upper respiratory tract, α2-6-linked SAs (the preferred receptor for human influenza viruses) predominate on epithelial cells (26). While α2-3-linked SAs are also present to a lesser degree on respiratory epithelial cells, this linkage is more abundantly expressed on secreted mucins (2). In contrast, α2-3-linked SAs (the preferred receptor for avian influenza viruses) are found on corneal and conjunctival epithelial cells of the human eye (31, 41), while secreted ocular mucins are abundantly composed of α2-6 SAs (5). It has been suggested that avian influenza viruses are more suited to infect the ocular surface due to their general α2-3-linked SA binding preference, but this has not been demonstrated experimentally (30).The mouse model has been used previously to study the role of ocular exposure to respiratory viruses (6, 39). In mice, ocular inoculation with an H3N2 influenza virus resulted in virus replication in nasal turbinates and lung (39), whereas ocular infection with respiratory syncytial virus (RSV) resulted in detectable virus titers in the eye and lung (6). These studies have revealed that respiratory viruses are not limited to the ocular area following inoculation at this site. However, the ability of influenza viruses to replicate specifically within ocular tissue has not been examined.Despite repeated instances of conjunctivitis associated with H7 subtype infections in humans, the reasons for this apparent ocular tropism have not been studied extensively. Here, we present a murine model to study the ability of human and avian influenza viruses to cause disease by the ocular route. We found that highly pathogenic H7 and H5 influenza viruses were capable of causing a systemic and lethal infection in mice following ocular inoculation. These highly pathogenic viruses, unlike human H3N2 and H1N1 viruses, replicated to significant titers in the mouse corneal epithelium and primary human corneal epithelial cells (HCEpiCs). Identification of viruses well suited to infecting the ocular surface is the first step in better understanding the ability of influenza viruses of multiple subtypes to use this tissue as a portal of entry.     

Evaluation of Recombinant Influenza Virus-Simian Immunodeficiency Virus Vaccines in Macaques

There is an urgent need for human immunodeficiency virus (HIV) vaccines that induce robust mucosal immunity. Influenza A viruses (both H1N1 and H3N2) were engineered to express simian immunodeficiency virus (SIV) CD8 T-cell epitopes and evaluated following administration to the respiratory tracts of 11 pigtail macaques. Influenza virus was readily detected from respiratory tract secretions, although the infections were asymptomatic. Animals seroconverted to influenza virus and generated CD8 and CD4 T-cell responses to influenza virus proteins. SIV-specific CD8 T-cell responses bearing the mucosal homing marker β7 integrin were induced by vaccination of naïve animals. Further, SIV-specific CD8 T-cell responses could be boosted by recombinant influenza virus-SIV vaccination of animals with already-established SIV infection. Sequential vaccination with influenza virus-SIV recombinants of different subtypes (H1N1 followed by H3N2 or vice versa) produced only a limited boost in immunity, probably reflecting T-cell immunity to conserved internal proteins of influenza A virus. SIV challenge of macaques vaccinated with an influenza virus expressing a single SIV CD8 T cell resulted in a large anamnestic recall CD8 T-cell response, but immune escape rapidly ensued and there was no impact on chronic SIV viremia. Although our results suggest that influenza virus-HIV vaccines hold promise for the induction of mucosal immunity to HIV, broader antigen cover will be needed to limit cytotoxic T-lymphocyte escape.Developing a safe and effective human immunodeficiency virus (HIV) vaccine is one of the defining scientific challenges of our time. Induction of peripheral CD8 T-cell immunity to HIV did not protect against sexual exposure to HIV type 1 (HIV-1) in humans in a recent efficacy trial (11, 43). In simian immunodeficiency virus (SIV)-macaque studies, peripheral CD8 T-cell immunity can effectively control viremia (40) but is often observed to have a transient or limited role in delaying SIV disease in macaques (32). The gradual accumulation of immune escape at CD8 T-cell epitopes undermines the effectiveness of CD8 T-cell immunity to SIV (6, 22, 46). It is likely that inducing mucosal CD8 T-cell immunity to HIV will be more effective at limiting viral replication during the very early phases of acute infection, prior to massive viral dissemination and destruction of large numbers of CD4 T cells (50). The induction of multifunctional mucosal CD8 T cells by live attenuated SIV vaccination of macaques is thought to play a significant role in the success of this strategy (25, 26); however, it is unfortunately too dangerous for clinical trials at present.A series of mucosal viral and bacterial HIV vaccine vectors have been studied in recent years; however, none have yet proceeded to advanced clinical trials. Live attenuated poliovirus vectors have shown promise in SIV studies, but these viruses can in rare cases revert to virulence (14). Salmonella-based SIV vaccine vectors are able to induce CD8 T-cell responses which express the α4β7 integrin mucosal homing marker when administered orally (20, 24). However, there may be a much stronger link between concomitant genital tract immunity and immunity induced at respiratory mucosal sites compared to that induced at enteric sites (33, 38, 42). Vesicular stomatitis virus vectors that replicate in the nasal mucosa show promise in SIV-macaque trials but are potentially neurotoxic (55). Replication-competent adenovirus vectors have looked promising in some SHIV-macaque studies (49) but failed to provide significant protection in a recent SIV-macaque study (17) and could have similar issues of enhanced infection rates as seen in the recent efficacy trials of replication-incompetent adenovirus type 5 vectors.A mucosal vector system that has several advantages over existing models but that is relatively unexplored is recombinant attenuated influenza viruses. Such viruses (i) have an existing reverse genetics system to readily generate and manipulate recombinant viruses (31, 34), (ii) are effective as anti-influenza vaccines and licensed for human use (e.g., “Flumist” vaccine [9]) with ready production capability, (iii) have robust respiratory mucosal replication that should facilitate genital mucosal immunity, and (iv) can be generated with a variety of hemagglutinin (H) and neuraminidase (N) glycoproteins, potentially enabling these viruses to be administered sequentially in prime-boost combinations to limit the effect of antivector humoral immunity (34). Mouse-adapted recombinant influenza virus-HIV vectors have been studied in mice and demonstrated significant induction of cellular immunity at mucosal sites (8, 27, 28, 44, 48). However, although several native influenza viruses replicate efficiently in the respiratory tracts of Asian macaque species (10, 12, 52), no studies to date have examined the immunogenicity or efficacy of recombinant attenuated influenza virus-SIV vectors in macaques.     

Utilization of Immunoglobulin G Fc Receptors by Human Immunodeficiency Virus Type 1: a Specific Role for Antibodies against the Membrane-Proximal External Region of gp41

Receptors (FcγRs) for the constant region of immunoglobulin G (IgG) are an important link between humoral immunity and cellular immunity. To help define the role of FcγRs in determining the fate of human immunodeficiency virus type 1 (HIV-1) immune complexes, cDNAs for the four major human Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa) were stably expressed by lentiviral transduction in a cell line (TZM-bl) commonly used for standardized assessments of HIV-1 neutralization. Individual cell lines, each expressing a different FcγR, bound human IgG, as evidence that the physical properties of the receptors were preserved. In assays with a HIV-1 multisubtype panel, the neutralizing activities of two monoclonal antibodies (2F5 and 4E10) that target the membrane-proximal external region (MPER) of gp41 were potentiated by FcγRI and, to a lesser extent, by FcγRIIb. Moreover, the neutralizing activity of an HIV-1-positive plasma sample known to contain gp41 MPER-specific antibodies was potentiated by FcγRI. The neutralizing activities of monoclonal antibodies b12 and 2G12 and other HIV-1-positive plasma samples were rarely affected by any of the four FcγRs. Effects with gp41 MPER-specific antibodies were moderately stronger for IgG1 than for IgG3 and were ineffective for Fab. We conclude that FcγRI and FcγRIIb facilitate antibody-mediated neutralization of HIV-1 by a mechanism that is dependent on the Fc region, IgG subclass, and epitope specificity of antibody. The FcγR effects seen here suggests that the MPER of gp41 could have greater value for vaccines than previously recognized.Fc receptors (FcRs) are differentially expressed on a variety of cells of hematopoietic lineage, where they bind the constant region of antibody (Ab) and provide a link between humoral and cellular immunity. Humans possess two classes of FcRs for the constant region of IgG (FcγRs) that, when cross-linked, are distinguished by their ability to either activate or inhibit cell signaling (69, 77, 79). The activating receptors FcγRI (CD64), FcγRIIa (CD32), and FcγRIII (CD16) signal through an immunoreceptor tyrosine-based activation motif (ITAM), whereas FcγRIIb (CD32) contains an inhibitory motif (ITIM) that counters ITAM signals and B-cell receptor signals. It has been suggested that a balance between activating and inhibitory FcγRs coexpressed on the same cells plays an important role in regulating adaptive immunity (23, 68). Moreover, the inhibitory FcγRIIb, being the sole FcγR on B cells, appears to play an important role in regulating self-tolerance (23, 68). The biologic role of FcγRs may be further influenced by differences in their affinity for immunoglobulin G (IgG); thus, FcγRI is a high-affinity receptor that binds monomeric IgG (mIgG) and IgG immune complexes (IC), whereas FcγRIIa, FcγRIIb, and FcγRIIIa are medium- to low-affinity receptors that preferentially bind IgG IC (10, 49, 78). FcγRs also exhibit differences in their relative affinity for the four IgG subclasses (10), which has been suggested to influence the balance between activating and inhibitory FcγRs (67).In addition to their participation in acquired immunity, FcγRs can mediate several innate immune functions, including phagocytosis of opsonized pathogens, Ab-dependent cell cytotoxicity (ADCC), antigen uptake by professional antigen-presenting cells, and the production of inflammatory cytokines and chemokines (26, 35, 41, 48, 69). In some cases, interaction of Ab-coated viruses with FcγRs may be exploited by viruses as a means to facilitate entry into FcγR-expressing cells (2, 33, 47, 84). Several groups have reported FcγR-mediated Ab-dependent enhancement (ADE) of HIV-1 infection in vitro (47, 51, 58, 63, 94, 96), whereas other reports have implicated FcγRs in efficient inhibition of the virus in vitro (19, 21, 29, 44-46, 62, 98) and possibly as having beneficial effects against HIV-1 in vivo (5, 27, 28, 42). These conflicting results are further complicated by the fact that HIV-1-susceptible cells, such as monocytes and macrophages, can coexpress more than one FcγR (66, 77, 79).HIV-1 entry requires sequential interactions between the viral surface glycoprotein, gp120, and its cellular receptor (CD4) and coreceptor (usually CCR5 or CXCR4), followed by membrane fusion that is mediated by the viral transmembrane glycoprotein gp41 (17, 106). Abs neutralize the virus by binding either gp120 or gp41 and blocking entry into cells. Several human monoclonal Abs that neutralize a broad spectrum of HIV-1 variants have attracted considerable interest for vaccine design. Epitopes for these monoclonal Abs include the receptor binding domain of gp120 in the case of b12 (71, 86), a glycan-specific epitope on gp120 in the case of 2G12 (13, 85, 86), and two adjacent epitopes in the membrane-proximal external region (MPER) of g41 in the cases of 2F5 and 4E10 (3, 11, 38, 93). At least three of these monoclonal Abs have been shown to interact with FcRs and to mediate ADCC (42, 43).A highly standardized and validated assay for neutralizing Abs against HIV-1 that quantifies reductions in luciferase (Luc) reporter gene expression after a single round of virus infection in TZM-bl cells has been developed (60, 104). TZM-bl (also called JC53BL-13) is a CXCR4-positive HeLa cell line that was engineered to express CD4 and CCR5 and to contain integrated reporter genes for firefly Luc and Escherichia coli β-galactosidase under the control of the HIV-1 Tat-regulated promoter in the long terminal repeat terminal repeat sequence (74, 103). TZM-bl cells are permissive to infection by a wide variety of HIV-1, simian immunodeficiency virus, and human-simian immunodeficiency virus strains, including molecularly cloned Env-pseudotyped viruses. Here we report the creation and characterization of four new TZM-bl cell lines, each expressing one of the major human FcγRs. These new cell lines were used to gain a better understanding of the individual roles that FcγRs play in determining the fate of HIV-1 IC. Two FcγRs that potentiated the neutralizing activity of gp41 MPER-specific Abs were identified.     

Mutations in the Bacillus subtilis β Clamp That Separate Its Roles in DNA Replication from Mismatch Repair

The β clamp is an essential replication sliding clamp required for processive DNA synthesis. The β clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of β clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49°C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures. We selected for intragenic suppressors of dnaN5 that rescued viability at 49°C to determine if the DNA replication defect could be separated from the MMR defect. We isolated three intragenic suppressors of dnaN5 that restored growth at the nonpermissive temperature while maintaining an increase in mutation frequency. All three dnaN alleles encoded the G73R substitution along with one of three novel missense mutations. The missense mutations isolated were S22P, S181G, and E346K. Of these, S181G and E346K are located near the hydrophobic cleft of the β clamp, a common site occupied by proteins that bind the β clamp. Using several methods, we show that the increase in mutation frequency resulting from each dnaN allele is linked to a defect in MMR. Moreover, we found that S181G and E346K allowed growth at elevated temperatures and did not have an appreciable effect on mutation frequency when separated from G73R. Thus, we found that specific residue changes in the B. subtilis β clamp separate the role of the β clamp in DNA replication from its role in MMR.Replication sliding clamps are essential cellular proteins imparting a spectacular degree of processivity to DNA polymerases during genome replication (24, 39-41). Encoded by the dnaN gene, the β clamp is a highly conserved bacterial sliding clamp found in virtually all eubacterial species (reviewed in reference 7). The β clamp is a head-to-tail, ring-shaped homodimer that encircles double-stranded DNA (1, 39). In eukaryotes and archaea, the analog of the β clamp is proliferating cell nuclear antigen (PCNA) (15, 28, 40, 41). Eukaryotic PCNA is a ring-shaped homotrimer that also acts to encircle DNA, increasing the processivity of the replicative DNA polymerases (40, 41). Although the primary structures of the β clamp and PCNA are not conserved, the tertiary structures of these proteins are very similar, demonstrating structural conservation among bacterial, archaeal, and eukaryotic replication sliding clamps (28, 39-41; reviewed in reference 6).The function of the β clamp is not limited to its well-defined role in genome replication. The Escherichia coli β clamp binds Hda, which also binds the replication initiation protein DnaA, regulating the active form of DnaA complexed with ATP (19, 37, 43). This allows the β clamp to regulate replication initiation through the amount of available DnaA-ATP. In Bacillus subtilis, the β clamp binds YabA, a negative regulator of DNA replication initiation (12, 29, 52). It has also been suggested that the B. subtilis β clamp sequesters DnaA from the replication origin during the cell cycle through the binding of DnaA to YabA and the binding of YabA to the β clamp (70). Thus, it is hypothesized that in E. coli and B. subtilis, the β clamp influences the frequency of replication initiation through interactions with Hda and YabA, respectively.The E. coli and B. subtilis β clamp has an important role in translesion DNA synthesis during the replicative bypass of noncoding bases by specialized DNA polymerases belonging to the Y family (20, 33). The roles of the E. coli β clamp in translesion synthesis are well established (5, 8, 30, 31). Binding sites on the E. coli β clamp that accommodate translesion polymerases pol IV (DinB) and pol V (UmuD₂′C) have been identified, and the consequence of disrupting their association with the β clamp has illustrated the critical importance of the β clamp to the activity of both of these polymerases (4, 5, 8, 26, 30, 31, 48, 49).In addition to the involvement of the β clamp in replication initiation, DNA replication, and translesion synthesis, the E. coli and B. subtilis β clamp also functions in DNA mismatch repair (MMR) (45, 46, 64). The MMR pathway recognizes and repairs DNA polymerase errors, contributing to the overall fidelity of the DNA replication pathway (reviewed in references 42 and 60). In both E. coli and B. subtilis, deletion of the genes mutS and mutL increases the spontaneous mutation frequency several hundredfold (13, 25, 63). In E. coli, MutS recognizes and binds mismatches, while MutL functions as a “matchmaker,” coordinating the actions of other proteins in the MMR pathway, allowing the removal of the mismatch and resynthesis of the resulting gap (reviewed in references 42 and 60). MutS and MutL of E. coli and B. subtilis physically interact with the β clamp (45, 46, 51, 64). Interaction between the B. subtilis β clamp and MutS is important for efficient MMR and organization of MutS-green fluorescent protein (GFP) into foci in response to replication errors, while the function of MutL binding to the β clamp is unknown (64).These studies show that the β clamp is critical for several aspects of DNA metabolism in E. coli and B. subtilis. In E. coli, many dnaN alleles have been examined and used to define the mechanistic roles of the β clamp in vivo (5, 18, 24, 30, 31, 48, 49, 73). A limitation in studying the mechanistic roles of the B. subtilis β clamp is that only two dnaN alleles (β clamp) are available, dnaN5 and dnaN34 (36) (www.bgsc.org/), and both of these alleles do not support growth at temperatures above 49°C, suggesting that they may cause similar defects (36) (www.bgsc.org/). Of these two dnaN alleles, only dnaN5 has been investigated in any detail (36, 53, 64). The mutant β clamp encoded by dnaN5 contains a G73R substitution [dnaN5(G73R)] in a surface-exposed residue located on the outside rim of the β clamp (53, 64). Our previous studies with this allele showed that dnaN5(G73R) confers an increase in mutation frequency at 30°C and 37°C (64). Further characterization of dnaN5(G73R) showed that the increased mutation frequency is caused by a partial defect in MMR (64). Additionally, dnaN5(G73R)-containing cells have a reduced ability to support MutS-GFP focus formation in response to mismatches (64). These results support the hypothesis that G73R in the β clamp causes a defect in DNA replication at 49°C (36) and impaired MMR manifested by a defect in establishing the assembly of MutS-GFP foci in response to replication errors (64).To understand the roles of the B. subtilis β clamp in MMR and DNA replication, we examined the dnaN5 and dnaN34 alleles. We found that the nucleotide sequences of dnaN5 and dnaN34 and the phenotypes they produce were identical, both producing the G73R missense mutation. We analyzed in vivo β clamp^G73R protein levels and found that the β clamp^G73R protein accumulated to wild-type levels at elevated temperatures. To identify amino acid residues that would restore DNA replication at elevated temperatures, we isolated three intragenic suppressors of dnaN5(G73R) that conferred growth of B. subtilis cells at 49°C. Epistasis analysis and determination of the mutation spectrum showed that each dnaN allele isolated in this study caused an MMR-dependent increase in mutation frequency. Additionally, we found that the β clamp binding protein YabA can reduce the efficiency of MMR in vivo when yabA expression is induced. Thus, we have identified residues in the β clamp that are critical for DNA replication and MMR in B. subtilis. We also found that a β clamp binding protein, YabA, can reduce the efficiency of MMR in vivo.     

Virulence-Associated Substitution D222G in the Hemagglutinin of 2009 Pandemic Influenza A(H1N1) Virus Affects Receptor Binding

The clinical impact of the 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively low. However, amino acid substitution D222G in the hemagglutinin of pdmH1N1 has been associated with cases of severe disease and fatalities. D222G was introduced in a prototype pdmH1N1 by reverse genetics, and the effect on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models was investigated. pdmH1N1 with D222G caused ocular disease in mice without further indications of enhanced virulence in mice and ferrets. pdmH1N1 with D222G retained transmissibility via aerosols or respiratory droplets in ferrets and guinea pigs. The virus displayed changes in attachment to human respiratory tissues in vitro, in particular increased binding to macrophages and type II pneumocytes in the alveoli and to tracheal and bronchial submucosal glands. Virus attachment studies further indicated that pdmH1N1 with D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids. Molecular dynamics modeling of the hemagglutinin structure provided an explanation for the retention of α2,6 binding. Altered receptor specificity of the virus with D222G thus affected interaction with cells of the human lower respiratory tract, possibly explaining the observed association with enhanced disease in humans.In April 2009, the H1N1 influenza A virus of swine origin was detected in humans in North America (9, 12, 42). Evidence for its origin came from analyses of the viral genome, with six gene segments displaying the closest resemblance to American “triple-reassortant” swine viruses and two to “Eurasian-lineage” swine viruses (13, 42). After this first detection in humans, the virus spread rapidly around the globe, starting the first influenza pandemic of the 21st century. The 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively mild, with a spectrum of disease ranging from subclinical infections or mild upper respiratory tract illness to sporadic cases of severe pneumonia and acute respiratory distress syndrome (3, 11, 27, 29, 30, 37). Overall, the case-fatality rate during the start of the pandemic was not significantly higher than in seasonal epidemics in most countries. However, a marked difference was observed in the case-fatality rate in specific age groups, with seasonal influenza generally causing highest mortality in elderly and immunocompromised individuals, and the pdmH1N1 affecting a relatively large proportion of (previously healthy) young individuals (3, 11, 27, 29, 30, 37).Determinants of influenza A virus virulence have been mapped for a wide variety of zoonotic and pandemic influenza viruses to the polymerase genes, hemagglutinin (HA), neuraminidase (NA), and nonstructural protein 1 (NS1). Such virulence-associated substitutions generally facilitate more efficient replication in humans via improved interactions with host cell factors. Since most of these virulence-associated substitutions were absent in the earliest pdmH1N1s, it has been speculated that the virus could acquire some of these mutations, potentially resulting in the emergence of more pathogenic viruses. Such virulence markers could be acquired by gene reassortment with cocirculating influenza A viruses, or by mutation. The influenza virus polymerase genes, in particular PB2, have been shown to be important determinants of the virulence of the highly pathogenic avian influenza (HPAI) H5N1 and H7N7 viruses and the transmission of the 1918 H1N1 Spanish influenza virus (17, 26, 34, 51). One of the most commonly identified virulence markers to date is E627K in PB2. The glutamic acid (E) residue is generally found in avian influenza viruses, while human viruses have a lysine (K), and this mutation was described as a determinant of host range in vitro (48). Given that all human and many zoonotic influenza viruses of the last century contained 627K, it was surprising that the pdmH1N1 had 627E. In addition, an aspartate (D)-to-asparagine (N) substitution at position 701 (D701N) of PB2 has previously been shown to expand the host range of avian H5N1 virus to mice and humans and to increase virus transmission in guinea pigs (26, 46). Like E627K, D701N was absent in the genome of pdmH1N1. Thus, the pdmH1N1 was the first known human pandemic virus with 627E and 701D, and it has been speculated that pdmH1N1 could mutate into a more virulent form by acquiring one of these mutations or both. Recently, it was shown that neither E627K nor D701N in PB2 of pdmH1N1 increased its virulence in ferrets and mice (18). The PB1-F2 protein has previously also been associated with high pathogenicity of the 1918 H1N1 and HPAI H5N1 viruses (8). The PB1-F2 protein of the pdmH1N1 is truncated due to premature stop codons. However, restoration of the PB1-F2 reading frame did not result in viruses with increased virulence (15). The NS1 protein of pdmH1N1 is also truncated due to a stop codon and, as a result, does not contain a PDZ ligand domain that is involved in cell-signaling pathways and has been implicated in the pathogenicity of 1918 H1N1 and HPAI H5N1 viruses (5, 8, 21). Surprisingly, restoration of a full-length version of the NS1 gene did not result in increased virulence in animal models (16). Mutations affecting virulence and host range have further frequently been mapped to hemagglutinin (HA) and neuraminidase (NA) in relation to their interaction with α2,3- or α2,6-linked sialic acids (SAs), the virus receptors on host cells (17, 32, 35, 50). The HA gene of previous pandemic viruses incorporated substitutions that allow efficient attachment to α2,6-SAs—the virus receptor on human cells—compared to ancestral avian viruses that attach more efficiently to α2,3-SAs (35, 47, 50).To search for mutations of potential importance to public health, numerous laboratories performed genome sequencing of pdmH1N1s, resulting in the real-time accumulation of information on emergence of potential virulence markers. Of specific interest were reports on amino acid substitutions from aspartic acid (D) to glycine (G) at position 222 (position 225 in H3) in HA of pdmH1N1. This substitution was observed in a fatal case of pdmH1N1 infection in June 2009 in the Netherlands (M. Jonges et al., unpublished data). Between July and December 2009, viruses from 11 (18%) of 61 cases with severe disease outcome in Norway have also been reported to harbor the D222G substitution upon direct sequencing of HA in clinical specimens. Such mutant viruses were not observed in any of 205 mild cases investigated, and the frequency of detection of this mutation was significantly higher in severe cases than in mild cases (23). In Hong Kong, the D222G substitution was detected in 12.5% (6) and 4.1% (31) of patients with severe disease and in 0% of patients with mild disease, in two different studies without prior propagation in embryonated chicken eggs. In addition to Norway and Hong Kong, the mutation has been detected in Brazil, Japan, Mexico, Ukraine, and the United States (56). Thus, D222G in HA could be the first identified “virulence marker” of pdmH1N1. pdmH1N1 with D222G in HA have not become widespread in the population, although they were detected in several countries. However, D222G in HA is of special interest, since it has also been described as the single change in HA between two strains of the “Spanish” 1918 H1N1 virus that differed in receptor specificity (47). Furthermore, upon propagation in embryonated chicken eggs, pdmH1N1 can acquire the mutation rapidly, presumably because it results in virus adaptation to avian (α2,3-SAs) receptors (49). The presence of the substitution in pdmH1N1s in the human population and its potential association with more severe disease prompted us to test its effect on pdmH1N1 receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models.     

A Basic Patch on α-Adaptin Is Required for Binding of Human Immunodeficiency Virus Type 1 Nef and Cooperative Assembly of a CD4-Nef-AP-2 Complex

A critical function of the human immunodeficiency virus type 1 Nef protein is the downregulation of CD4 from the surfaces of infected cells. Nef is believed to act by linking the cytosolic tail of CD4 to the endocytic machinery, thereby increasing the rate of CD4 internalization. In support of this model, weak binary interactions between CD4, Nef, and the endocytic adaptor complex, AP-2, have been reported. In particular, dileucine and diacidic motifs in the C-terminal flexible loop of Nef have been shown to mediate binding to a combination of the α and σ2 subunits of AP-2. Here, we report the identification of a potential binding site for the Nef diacidic motif on α-adaptin. This site comprises two basic residues, lysine-297 and arginine-340, on the α-adaptin trunk domain. The mutation of these residues specifically inhibits the ability of Nef to bind AP-2 and downregulate CD4. We also present evidence that the diacidic motif on Nef and the basic patch on α-adaptin are both required for the cooperative assembly of a CD4-Nef-AP-2 complex. This cooperativity explains how Nef is able to efficiently downregulate CD4 despite weak binary interactions between components of the tripartite complex.CD4, a type I transmembrane glycoprotein that serves as a coreceptor for major histocompatibility complex class II (MHC-II) molecules, is expressed on the surfaces of helper T lymphocytes and cells of the monocyte/macrophage lineage (8). Primate immunodeficiency viruses gain access to these cells by virtue of the interaction of the viral envelope glycoprotein (Env) with a combination of CD4 and a chemokine receptor (63). This interaction causes a conformational change within the Env protein that promotes the fusion of the viral envelope with the plasma membrane. Upon the delivery of the viral genetic material into the cytoplasm of the host cells, one of the first virally encoded proteins to be expressed is Nef, an accessory factor that modulates specific signal transduction and protein-trafficking pathways in a manner that optimizes the intracellular environment for viral replication (reviewed in references 21, 39, and 65). Perhaps the best characterized function of Nef is the downregulation of CD4 from the surfaces of the host cells (6, 22, 29, 45). CD4 downregulation prevents superinfection (6, 41) and enhances virion release (19, 38, 48, 66, 76), thereby contributing to the establishment of a robust infective state (24, 72).The mechanism used by the Nef protein of human immunodeficiency virus type 1 (HIV-1) to downregulate CD4 has been the subject of extensive study, but only recently have the molecular details of this process begun to be unraveled. It is generally acknowledged that HIV-1 Nef accelerates the internalization of CD4 from the plasma membrane by linking the cytosolic tail of the receptor to the clathrin-associated endocytic machinery (1, 12, 20, 34, 40, 64). In support of this model, a hydrophobic pocket comprising W57 and L58 on the folded core domain of Nef binds with millimolar affinity to the cytosolic tail of CD4 (28) (all residues and numbers correspond to the NL4-3 variant of HIV-1 Nef used in this study). In addition, a dileucine motif (ENTSLL, residues 160 to 165) (10, 16, 26) and a diacidic motif (D174 and D175) (2) on the C-terminal flexible loop of Nef mediate an interaction of micromolar affinity with the clathrin-associated, heterotetrameric (α-β2-μ2-σ2) adaptor protein 2 (AP-2) complex (12, 20, 40, 49). These interactions draw CD4 into clathrin-coated pits that eventually bud inwards as clathrin-coated vesicles (11, 27). Internalized CD4 is subsequently delivered to endosomes and then to lysosomes for degradation (3, 23, 59, 64).Despite progress in the understanding of the mechanism of Nef-induced CD4 downregulation, several important aspects remain to be elucidated. Previous studies have shown that the Nef dileucine and diacidic motifs interact with a combination of the α and σ2 subunits of AP-2 (referred to as the α-σ2 hemicomplex) (12, 20, 40, 49), but the precise location of the Nef binding sites is unknown. It also remains to be determined whether Nef can actually bind CD4 and AP-2 at the same time. Indeed, the formation of a tripartite CD4-Nef-AP-2 complex in which Nef links the cytosolic tail of CD4 to AP-2 has long been hypothesized but has never been demonstrated experimentally. Given the relatively weak affinity of Nef for the CD4 tail (28) and AP-2 (12, 40), it is unclear how such a complex could assemble and function in CD4 downregulation.In this study, we have addressed these issues by using a combination of yeast hybrid, in vitro binding, and in vivo CD4 downregulation assays. We report the identification of a candidate binding site for the Nef diacidic motif on the AP-2 complex. This site, a basic patch comprising K297 and R340 on α-adaptin, is specifically required for Nef binding and Nef-induced CD4 downregulation. We also show that the Nef diacidic motif and the α-adaptin basic patch are required for the cooperative assembly of a tripartite complex composed of the CD4 cytosolic tail, Nef, and the α-σ2 hemicomplex. The cooperative manner in which this complex is formed explains how Nef is able to efficiently downregulate CD4 from the plasma membrane despite weak binary interactions between the components of this complex.