Influenza infection causes airway hyperresponsiveness by decreasing enkephalinase

Ferret tracheal segments were infected with human influenza virus A/Taiwan/86 (H1N1) in vitro. After 4 days, the smooth muscle contractile responses to acetylcholine and to substance P were measured. The response to substance P was markedly accentuated, with a threefold increase in force of contraction at a substance P concentration of 10(-5) M, the highest concentration tested. In contrast, the response to acetylcholine was not affected by viral infection. Histological examination of tissues revealed extensive epithelial desquamation. Activity of enkephalinase (neutral metallo-endopeptidase, EC.3.4.24.11), an enzyme that degrades substance P, was decreased by 50% in infected tissues. Inhibiting enkephalinase activity by pretreating with thiorphan (10(-5) M) increased the response to substance P to the same final level in both infected and control tissues. Inhibiting other substance P-degrading enzymes including kininase II (angiotensin-converting enzyme), serine proteases, and aminopeptidases did not affect the response to substance P. Inhibiting cyclooxygenase and lipoxygenase activity using indomethacin and BW 755c did not affect hyperresponsiveness to substance P. Pretreating tissues with antagonists of alpha-adrenoceptors, beta-adrenoceptors, and H1 histamine receptors (phentolamine 10(-5) M, propranolol 5 X 10(-6) M, and pyrilamine 10(-5) M, respectively) had no effect on substance P-induced contraction. These results demonstrate that infection of ferret airway tissues with influenza virus increases the contractile response of airway smooth muscle to substance P. This effect is caused by decreased enkephalinase activity in infected tissues.     

Effect of Staphylococcus enterotoxin B on the concurrent CD8(+) T cell response to influenza virus infection

Bacterial superantigens have potent in vivo effects. Respiratory viral infections are often associated with secondary bacterial infections, raising the likelihood of exposure to bacterial superantigens after the initiation of the anti-viral immune response. In this study, the general and V beta-specific effects of exposure to Staphylococcal enterotoxin B (SEB) during influenza virus infection on both the ongoing acute and the subsequent recall CD8(+) T cell responses were analyzed, using the well-characterized murine influenza model system and tetrameric MHC/peptide reagents to directly identify virus-specific T cells. The results show that although superantigen exposure during the primary viral infection caused delayed viral clearance, there was remarkably little effect of SEB on the magnitude or TCR repertoire of the ongoing cytolytic T cell response or on the recall response elicited by secondary viral infection. Thus, despite the well-characterized immunomodulatory effects of SEB, there was surprisingly little interference with concurrent anti-viral immunity.     

Modulation of the contractile responses of guinea pig isolated tracheal rings after chronic intermittent hypobaric hypoxia with and without cold exposure.

Previous studies have documented that repetitive exposure to intermittent hypoxia, such as that encountered in preparation to high-altitude ascent, influences breathing. However, the impact of intermittent hypoxia on airway smooth muscle has not been explored. Ascents to high altitude, in addition to hypoxia, expose individuals to cold air. The objective of the present study is to examine the effect of chronic intermittent hypobaric hypoxia (CIH) and CIH combined with cold exposure (CIHC) on tracheal smooth muscle responses to various contractile and relaxant agonists. Experiments were performed on tracheal rings harvested from adult guinea pigs exposed either to CIH or CIHC [14 days (6 h/day) at barometric pressure of 350 mmHg with and without cold exposure of 5 degrees C] or to room air (normoxia). CIH and CIHC attenuated maximum contractile responses to ACh compared with normoxia. The maximum contractile response to histamine decreased with CIH, whereas CIHC restored the response back to normoxia. Both CIH and CIHC attenuated maximum contractile responses to 5-HT. Altered contractile responses after CIH and CIHC were independent of epithelium. Isoproterenol-induced relaxation was not altered by CIH, whereas it was enhanced after CIHC, and these responses were independent of the epithelium. The data demonstrate that intermittent exposure to hypoxia profoundly influences contractile response of tracheal smooth muscle, and cold exposure can further modulate the response, implying the importance of cold at high altitude.     

Autocrine cytokine signaling mediates effects of rhinovirus on airway responsiveness

The airway responses to allergen exposure in allergic asthma are qualitatively similar to those elicited by specific viral respiratory pathogens, most notably rhinovirus (RV), suggesting that the altered airway responsiveness seen in allergic asthma and that elicited by viral respiratory tract infection may share a common underlying mechanism. To the extent that T helper cell type 2 (Th2) cytokines have been implicated in the pathogenesis of allergic asthma, this study examined the potential role(s) of Th2-type cytokines in mediating pro-asthmatic-like changes in airway smooth muscle (ASM) responsiveness after inoculation of naive ASM with human RV. Isolated rabbit ASM tissues and cultured human ASM cells were exposed to RV (serotype 16) for 24 h in the absence and presence of monoclonal blocking antibodies (MAbs) or antagonists directed against either the Th2-type cytokines interleukin (IL)-4 and IL-5, intercellular adhesion molecule (ICAM)-1 (the endogenous host receptor for most RVs), or the pleiotropic proinflammatory cytokine IL-1beta. Relative to control (vehicle-treated) tissues, RV-exposed ASM exhibited significantly enhanced isometric contractility to acetylcholine and impaired relaxation to isoproterenol. These pro-asthmatic-like changes in ASM responsiveness were ablated by pretreating the RV-exposed tissues with either IL-5-receptor-alpha blocking antibody or human recombinant IL-1-receptor antagonist, whereas IL-4 neutralizing antibody had no effect. Extended studies further demonstrated that inoculation of ASM cells with RV elicited 1) an increased mRNA expression and release of IL-5 protein, which was inhibited in the presence of anti-ICAM-1 MAb, and 2) an enhanced release of IL-1beta protein, which was inhibited in the presence of IL-5 receptor-alpha antibody. Collectively, these observations provide new evidence demonstrating that RV-induced changes in ASM responsiveness are largely attributed to ICAM-1-dependent activation of a cooperative autocrine signaling mechanism involving upregulated IL-5-mediated release of IL-1beta by the RV-exposed ASM itself.     

Homo- and heterosubtypic low pathogenic avian influenza exposure on H5N1 highly pathogenic avian influenza virus infection in wood ducks (Aix sponsa)

Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoirs for avian influenza (AI) viruses. Although they are often infected with multiple AI viruses, the significance and extent of acquired immunity in these populations is not understood. Pre-existing immunity to AI virus has been shown to modulate the outcome of a highly pathogenic avian influenza (HPAI) virus infection in multiple domestic avian species, but few studies have addressed this effect in wild birds. In this study, the effect of pre-exposure to homosubtypic (homologous hemagglutinin) and heterosubtypic (heterologous hemagglutinin) low pathogenic avian influenza (LPAI) viruses on the outcome of a H5N1 HPAI virus infection in wood ducks (Aix sponsa) was evaluated. Pre-exposure of wood ducks to different LPAI viruses did not prevent infection with H5N1 HPAI virus, but did increase survival associated with H5N1 HPAI virus infection. The magnitude of this effect on the outcome of the H5N1 HPAI virus infection varied between different LPAI viruses, and was associated both with efficiency of LPAI viral replication in wood ducks and the development of a detectable humoral immune response. These observations suggest that in naturally occurring outbreaks of H5N1 HPAI, birds with pre-existing immunity to homologous hemagglutinin or neuraminidase subtypes of AI virus may either survive H5N1 HPAI virus infection or live longer than naïve birds and, consequently, could pose a greater risk for contributing to viral transmission and dissemination. The mechanisms responsible for this protection and/or the duration of this immunity remain unknown. The results of this study are important for surveillance efforts and help clarify epidemiological data from outbreaks of H5N1 HPAI virus in wild bird populations.     

Mast cell-induced lung injury in mice infected with H5N1 influenza virus

Although an important role for mast cells in several viral infections has been demonstrated, its role in the invasion of highly pathogenic H5N1 influenza virus is unknown. In the present study, we demonstrate that mast cells were activated significantly by H5N1 virus (A/chicken/Henan/1/2004) infection both in vivo and in vitro. Mast cells could possibly intensify the lung injury that results from H5N1 infection by releasing proinflammatory mediators, including histamine, tryptase, and gamma interferon (IFN-γ). Lung lesions and apoptosis induced by H5N1 infection were reduced dramatically by treatment with ketotifen, which is a mast cell degranulation inhibitor. A combination of ketotifen and the neuraminidase inhibitor oseltamivir protected 100% of the mice from death postinfection. In conclusion, our data suggest that mast cells play a crucial role in the early stages of H5N1 influenza virus infection and provide a new approach to combat highly pathogenic influenza virus infection.     

Viral replication and innate host responses in primary human alveolar epithelial cells and alveolar macrophages infected with influenza H5N1 and H1N1 viruses

Highly pathogenic influenza H5N1 virus continues to pose a threat to public health. Although the mechanisms underlying the pathogenesis of the H5N1 virus have not been fully defined, it has been suggested that cytokine dysregulation plays an important role. As the human respiratory epithelium is the primary target cell for influenza viruses, elucidating the viral tropism and innate immune responses of influenza H5N1 virus in the alveolar epithelium may help us to understand the pathogenesis of the severe pneumonia associated with H5N1 disease. Here we used primary cultures of differentiated human alveolar type II cells, alveolar type I-like cells, and alveolar macrophages isolated from the same individual to investigate viral replication competence and host innate immune responses to influenza H5N1 (A/HK/483/97) and H1N1 (A/HK/54/98) virus infection. The viral replication kinetics and cytokine and chemokine responses were compared by quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). We demonstrated that influenza H1N1 and H5N1 viruses replicated productively in type II cells and type I-like cells although with different kinetics. The H5N1 virus replicated productively in alveolar macrophages, whereas the H1N1 virus led to an abortive infection. The H5N1 virus was a more potent inducer of proinflammatory cytokines and chemokines than the H1N1 virus in all cell types. However, higher levels of cytokine expression were observed for peripheral blood monocyte-derived macrophages than for alveolar macrophages in response to H5N1 virus infection. Our findings provide important insights into the viral tropisms and host responses of different cell types found in the lung and are relevant to an understanding of the pathogenesis of severe human influenza disease.     

Respiratory infection with influenza A virus interferes with the induction of tolerance to aeroallergens

Viral respiratory infections have been implicated in influencing allergen sensitization and the development of asthma, but their exact role remains controversial. Because respiratory exposure to Ag normally engenders T cell tolerance and prevents the development of airway hyperreactivity (AHR) and inflammation, we examined the effects of influenza A virus infection on tolerance induced by exposure to intranasal (i.n.) OVA and the subsequent development of AHR. We found that concurrent infection with influenza A abrogated tolerance induced by exposure to i.n. OVA, and instead led to the development of AHR accompanied by the production of OVA-specific IgE, IL-4, IL-5, IL-13, and IFN-gamma. When both IL-4 and IL-5 were neutralized in this system, AHR was still induced, suggesting that influenza-induced cytokines such as IL-13, or mechanisms unrelated to cytokines, might be responsible for the development of AHR. The length of time between influenza A infection and i.n. exposure to OVA was crucial, because mice exposed to i.n. OVA 15-30 days after viral inoculation developed neither AHR nor OVA-specific tolerance. These mice instead acquired Th1-biased OVA-specific immune responses associated with vigorous OVA-induced T cell proliferation, and reduced production of OVA-specific IgE. The protective effect of influenza A on AHR was dependent on IFN-gamma, because protection was abrogated with a neutralizing anti-IFN-gamma mAb. These results suggest that viral respiratory infection interferes with the development of respiratory allergen-induced tolerance, and that the time interval between viral infection and allergen exposure is critical in determining whether viral infection will enhance, or protect against, the development of respiratory allergen sensitization and AHR.     

Influenza A viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (IRE1) stress pathway

Known therapies for influenza A virus infection are complicated by the frequent emergence of resistance. A therapeutic strategy that may escape viral resistance is targeting host cellular mechanisms involved in viral replication and pathogenesis. The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a primitive, evolutionary conserved molecular signaling cascade that has been implicated in multiple biological phenomena including innate immunity and the pathogenesis of certain viral infections. We investigated the effect of influenza A viral infection on ER stress pathways in lung epithelial cells. Influenza A virus induced ER stress in a pathway-specific manner. We showed that the virus activates the IRE1 pathway with little or no concomitant activation of the PERK and the ATF6 pathways. When we examined the effects of modulating the ER stress response on the virus, we found that the molecular chaperone tauroursodeoxycholic acid (TUDCA) significantly inhibits influenza A viral replication. In addition, a specific inhibitor of the IRE1 pathway also blocked viral replication. Our findings constitute the first evidence that ER stress plays a role in the pathogenesis of influenza A viral infection. Decreasing viral replication by modulating the host ER stress response is a novel strategy that has important therapeutic implications.     

Parasite-mediated upregulation of NK cell-derived gamma interferon protects against severe highly pathogenic H5N1 influenza virus infection

Outbreaks of influenza A viruses are associated with significant human morbidity worldwide. Given the increasing resistance to the available influenza drugs, new therapies for the treatment of influenza virus infection are needed. An alternative approach is to identify products that enhance a protective immune response. In these studies, we demonstrate that infecting mice with the Th1-inducing parasite Toxoplasma gondii prior to highly pathogenic avian H5N1 influenza virus infection led to decreased lung viral titers and enhanced survival. A noninfectious fraction of T. gondii soluble antigens (STAg) elicited an immune response similar to that elicited by live parasites, and administration of STAg 2 days after H5N1 influenza virus infection enhanced survival, lowered viral titers, and reduced clinical disease. STAg administration protected H5N1 virus-infected mice lacking lymphocytes, suggesting that while the adaptive immune response was not required for enhanced survival, it was necessary for STAg-mediated viral clearance. Mechanistically, we found that administration of STAg led to increased production of gamma interferon (IFN-γ) from natural killer (NK) cells, which were both necessary and sufficient for survival. Further, administration of exogenous IFN-γ alone enhanced survival from H5N1 influenza virus infection, although not to the same level as STAg treatment. These studies demonstrate that a noninfectious T. gondii extract enhances the protective immune response against severe H5N1 influenza virus infections even when a single dose is administered 2 days postinfection.     

Influenza virus infection is not affected by serum amyloid P component

BACKGROUND: Binding of serum amyloid P component (SAP) to its ligands, including bacteria, chromatin and amyloid fibrils, protects them from degradation, is anti-opsonic and anti-immunogenic. SAP thereby enhances the virulence of pathogenic bacteria to which it binds. However SAP also contributes to host resistance against bacteria to which it does not bind. Human SAP has been reported to bind to the influenza virus and inhibit viral invasion of cells in tissue culture. We therefore investigated a possible role of SAP in either host resistance or viral virulence during influenza infection in vivo. MATERIALS AND METHODS: The clinical course of mouse adapted influenza virus infection, the host antibody response, and viral replication, were compared in wild type mice, mice with targeted deletion of the SAP gene, and mice transgenic for human SAP. The effects of reconstitution of SAP deficient mice with pure human SAP, and of a drug that specifically blocks SAP binding in vivo, were also studied. Binding of mouse and human SAP to immobilized influenza virus was compared. RESULTS: The presence, absence, or availability for binding of SAP in vivo had no significant or consistent effect on the course or outcome of influenza infection, or on either viral replication or the anti-viral antibody response. Mouse SAP bound much less avidly than human SAP to influenza virus. CONCLUSIONS: In marked contrast to the dramatic effects of SAP deficiency on host resistance to different bacterial infections, mouse SAP apparently plays no significant role during infection of mice with influenza virus. Human SAP binds much more avidly than mouse SAP to the virus, but also had no effect on any of the parameters measured and is therefore unlikely to be involved in human influenza infection.     

Stiffness changes in cultured airway smooth muscle cells

Airwaysmooth muscle (ASM) cells in culture stiffen when exposed tocontractile agonists. Such cell stiffening may reflect activation ofthe contractile apparatus as well as polymerization of cytoskeletalbiopolymers. Here we have assessed the relative contribution of thesemechanisms in cultured ASM cells stimulated with serotonin(5-hydroxytryptamine; 5-HT) in the presence or absence of drugs thatinhibit either myosin-based contraction or polymerization offilamentous (F) actin. Magnetic twisting cytometry was used to measurecell stiffness, and associated changes in structural organization ofactin cytoskeleton were evaluated by confocal microscopy. We found that5-HT increased cell stiffness in a dose-dependent fashion and alsoelicited rapid formation of F-actin as marked by increased intensity ofFITC-phalloidin staining in these cells. A calmodulin antagonist (W-7),a myosin light chain kinase inhibitor (ML-7) and a myosin ATPaseinhibitor (BDM) each ablated the stiffening response but not theF-actin polymerization induced by 5-HT. Agents that inhibited theformation of F-actin (cytochalasin D, latrunculin A, C3 exoenzyme, andY-27632) attenuated both baseline stiffness and the extent of cellstiffening in response to 5-HT. Together, these data suggest thatagonist-evoked stiffening of cultured ASM cells requires actinpolymerization as well as myosin activation and that neitheractin polymerization nor myosin activation by itself is sufficient toaccount for the cell stiffening response.

     

The inhibitory actions of acebutolol and propranolol on the contractile response to 5-hydroxytryptamine in various isolated vascular smooth muscles

Pretreatment with acebutolol or propranolol at high concentrations had an inhibitory effect on the contractile response to 5-hydroxytryptamine (5-HT) in most vascular smooth muscles such as rabbit aorta and basilar, mesenteric, renal, femoral arteries and cat coronary artery. The inhibitory actions of both agents were generally greater than on the responses to excess Ca2+ and potassium. In rabbit renal arteries, acebutolol had no effect on the response to 5-HT but inhibited the responses to excess Ca2+ and potassium. Propranolol had a marked inhibitory effect on the response to 5-HT. In all preparations used, the contractions induced by norepinephrine (NE) and histamine showed a much greater resistance to the effect of acebutolol and propranolol than the contractions induced by 5-HT, Ca2+ and potassium. Nifedipine had no inhibitory effect on the response to 5-HT in most of the preparations. Nifedipine inhibited the response to 5-HT only in the basilar arteries. The inhibitory actions of propranolol on the response to 5-HT was greater than that of acebutolol. The inhibitory action of acebutolol and propranolol on the response to 5-HT may be related to mechanisms other than the beta-adrenoceptor blocking action of the drugs. The possible mechanisms of inhibitory action of both beta-adrenoceptor antagonists on 5-HT are discussed.     

Respiratory epithelium-dependent inhibition of protein kinase C of airway smooth muscle cells

We have examined the effect of phorbol myristate acetate (PMA) on airway smooth muscle (ASM) in the presence and absence of respiratory epithelium (RE) and analyzed the dependence of this response on extracellular sodium, Na+/H+ exchange, calcium, and cyclooxygenase products; we determined both the resting membrane potential and isometric force developed by ASM preparations. Removal of RE had no effect on the values of the resting membrane potential of ASM cells. In the presence of RE in the preparation, both electrical and contractile responses to PMA (10(-5) M) were significantly different compared with the response of ASM to PMA without RE. When the RE was present, stimulation of protein kinase C caused only a biphasic response in both membrane potential and isometric force. In either the presence or absence of RE, amiloride (10(-5) M) and a low-sodium solution inhibited both electrical and contractile changes of ASM cells caused by PMA. In the presence or absence of RE, verapamil (10(-5) M) attenuated (P less than 0.05) both electrical and contractile responses of ASM cells as induced by PMA. Verapamil, however, had no effect on the last phase of PMA-induced response. Pretreatment of preparations with indomethacin (10(-6) M) changed the PMA-induced response of ASM with RE to a response usually observed in ASM without RE. Finally, the incubation of tracheal preparations without RE with prostaglandin E2 (10(-8) M) altered the response of these preparations in such a way that their electrical and contractile response to PMA was essentially identical to the PMA response observed in preparations with an intact RE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Critical role of constitutive type I interferon response in bronchial epithelial cell to influenza infection

Innate antiviral responses in bronchial epithelial cells (BECs) provide the first line of defense against respiratory viral infection and the effectiveness of this response is critically dependent on the type I interferons (IFNs). However the importance of the antiviral responses in BECs during influenza infection is not well understood. We profiled the innate immune response to infection with H3N2 and H5N1 virus using Calu-3 cells and primary BECs to model proximal airway cells. The susceptibility of BECs to influenza infection was not solely dependent on the sialic acid-bearing glycoprotein, and antiviral responses that occurred after viral endocytosis was more important in limiting viral replication. The early antiviral response and apoptosis correlated with the ability to limit viral replication. Both viruses reduced RIG-I associated antiviral responses and subsequent induction of IFN-β. However it was found that there was constitutive release of IFN-β by BECs and this was critical in inducing late antiviral signaling via type I IFN receptors, and was crucial in limiting viral infection. This study characterizes anti-influenza virus responses in airway epithelial cells and shows that constitutive IFN-β release plays a more important role in initiating protective late IFN-stimulated responses during human influenza infection in bronchial epithelial cells.     

Innate Immunity and the Inter-exposure Interval Determine the Dynamics of Secondary Influenza Virus Infection and Explain Observed Viral Hierarchies

Influenza is an infectious disease that primarily attacks the respiratory system. Innate immunity provides both a very early defense to influenza virus invasion and an effective control of viral growth. Previous modelling studies of virus–innate immune response interactions have focused on infection with a single virus and, while improving our understanding of viral and immune dynamics, have been unable to effectively evaluate the relative feasibility of different hypothesised mechanisms of antiviral immunity. In recent experiments, we have applied consecutive exposures to different virus strains in a ferret model, and demonstrated that viruses differed in their ability to induce a state of temporary immunity or viral interference capable of modifying the infection kinetics of the subsequent exposure. These results imply that virus-induced early immune responses may be responsible for the observed viral hierarchy. Here we introduce and analyse a family of within-host models of re-infection viral kinetics which allow for different viruses to stimulate the innate immune response to different degrees. The proposed models differ in their hypothesised mechanisms of action of the non-specific innate immune response. We compare these alternative models in terms of their abilities to reproduce the re-exposure data. Our results show that 1) a model with viral control mediated solely by a virus-resistant state, as commonly considered in the literature, is not able to reproduce the observed viral hierarchy; 2) the synchronised and desynchronised behaviour of consecutive virus infections is highly dependent upon the interval between primary virus and challenge virus exposures and is consistent with virus-dependent stimulation of the innate immune response. Our study provides the first mechanistic explanation for the recently observed influenza viral hierarchies and demonstrates the importance of understanding the host response to multi-strain viral infections. Re-exposure experiments provide a new paradigm in which to study the immune response to influenza and its role in viral control.     

Pulmonary vascular responsiveness in cold-exposed calves

The pulmonary vascular responses to acute hypoxia and to infusions of histamine and 5-hydroxytryptamine (5-HT) were recorded in unanesthetized standing bull calves under neutral (16-18 degrees C) and cold (3-5 degrees C) temperature conditions. Cold exposure alone resulted in a significant increase in pulmonary arterial wedge pressure from 10.2 +/- 3.5 to 15.9 +/- 4.9 Torr (1 Torr = 133.322 Pa). Resistance to blood flow between the pulmonary wedge and the left atrium significantly increased from 0.50 +/- 0.51 to 1.21 +/- 0.78 mmHg . L-1 . min-1 (1 mmHg = 133.322 Pa) with cold exposure. This apparent pulmonary venoconstrictor response to cold exposure was further evaluated to determine if hypoxia, histamine, or 5-HT responsiveness was altered by cold exposure. Twelve minutes of hypoxia increased pulmonary arterial and systemic arterial pressures, heart rate, and respiratory rate similarly in cold and neutral temperatures. Cold exposure did not alter the dose-related reductions of systemic arterial and pulmonary arterial pressures in response to histamine. Similarly, the decreases in systemic arterial pressure and heart rate and increases in pulmonary arterial and left atrial pressures in response to 5-HT were not significantly different in cold and neutral conditions. It was concluded that acute, mild cold exposure results in an increase in resistance to blood flow in the pulmonary venous circulation without a general increase in pulmonary vascular reactivity, as measured by responses to hypoxia, histamine, and 5-HT.     

Immunization of Pigs with a Particle-Mediated DNA Vaccine to Influenza A Virus Protects against Challenge with Homologous Virus

Particle-mediated delivery of a DNA expression vector encoding the hemagglutinin (HA) of an H1N1 influenza virus (A/Swine/Indiana/1726/88) to porcine epidermis elicits a humoral immune response and accelerates the clearance of virus in pigs following a homotypic challenge. Mucosal administration of the HA expression plasmid elicits an immune response that is qualitatively different than that elicited by the epidermal vaccination in terms of inhibition of the initial virus infection. In contrast, delivery of a plasmid encoding an influenza virus nucleoprotein from A/PR/8/34 (H1N1) to the epidermis elicits a strong humoral response but no detectable protection in terms of nasal virus shed. The efficacy of the HA DNA vaccine was compared with that of a commercially available inactivated whole-virus vaccine as well as with the level of immunity afforded by previous infection. The HA DNA and inactivated viral vaccines elicited similar protection in that initial infection was not prevented, but subsequent amplification of the infection is limited, resulting in early clearance of the virus. Convalescent animals which recovered from exposure to virulent swine influenza virus were completely resistant to infection when challenged. The porcine influenza A virus system is a relevant preclinical model for humans in terms of both disease and gene transfer to the epidermis and thus provides a basis for advancing the development of DNA-based vaccines.