A20 (Tnfaip3) Deficiency in Myeloid Cells Protects against Influenza A Virus Infection

The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules. Influenza A virus (IAV) produces double-stranded RNA as an intermediate during the replication life cycle, which activates the intracellular pathogen recognition receptor RIG-I and induces the production of proinflammatory cytokines and antiviral interferon. Understanding the mechanisms that regulate innate immune responses to IAV and other viruses is of key importance to develop novel therapeutic strategies. Here we used myeloid cell specific A20 knockout mice to examine the role of the ubiquitin-editing protein A20 in the response of myeloid cells to IAV infection. A20 deficient macrophages were hyperresponsive to double stranded RNA and IAV infection, as illustrated by enhanced NF-κB and IRF3 activation, concomitant with increased production of proinflammatory cytokines, chemokines and type I interferon. In vivo this was associated with an increased number of alveolar macrophages and neutrophils in the lungs of IAV infected mice. Surprisingly, myeloid cell specific A20 knockout mice are protected against lethal IAV infection. These results challenge the general belief that an excessive host proinflammatory response is associated with IAV-induced lethality, and suggest that under certain conditions inhibition of A20 might be of interest in the management of IAV infections.     

Augmented Lung Inflammation Protects against Influenza A Pneumonia

Background

Influenza pneumonia causes high mortality every year, and pandemic episodes kill millions of people. Influenza-related mortality has been variously ascribed to an ineffective host response that fails to limit viral replication, an excessive host inflammatory response that results in lung injury and impairment of gas exchange, or to bacterial superinfection. We sought to determine whether lung inflammation promoted or impaired host survival in influenza pneumonia.

Methods and Findings

To distinguish among these possible causes of influenza-related death, we induced robust lung inflammation by exposing mice to an aerosolized bacterial lysate prior to challenge with live virus. The treatment induced expression of the inflammatory cytokines IL-6 and TNF in bronchoalveolar lavage fluid 8- and 40-fold greater, respectively, than that caused by lethal influenza infection. Yet, this augmented inflammation was associated with striking resistance to host mortality (0% vs 90% survival, p = 0.0001) and reduced viral titers (p = 0.004). Bacterial superinfection of virus infected lungs was not observed. When mice were repeatedly exposed to the bacterial lysate, as would be clinically desirable during an influenza epidemic, there was no tachyphylaxis of the induced viral resistance. When the bacterial lysate was administered after the viral challenge, there was still some mortality benefit, and when ribavirin was added to the aerosolized bacterial lysate, host survival was synergistically improved (0% vs 93.3% survival, p<0.0001).

Conclusions

Together, these data indicate that innate immune resistance to influenza can be effectively stimulated, and suggest that ineffective rather than excessive inflammation is the major cause of mortality in influenza pneumonia.     

Lung Epithelial Cells Coordinate Innate Lymphocytes and Immunity against Pulmonary Fungal Infection

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Gastrointestinal Delivery of Baculovirus Displaying Influenza Virus Hemagglutinin Protects Mice against Heterologous H5N1 Infection

The recent outbreaks of influenza A H5N1 virus in birds and humans have necessitated the development of potent H5N1 vaccines. In this study, we evaluated the protective potential of an immediate-early promoter-based baculovirus displaying hemagglutinin (BacHA) against highly pathogenic avian influenza (HPAI) H5N1 virus infection in a mouse model. Gastrointestinal delivery of BacHA significantly enhanced the systemic immune response in terms of HA-specific serum IgG and hemagglutination inhibition (HI) titers. In addition, BacHA vaccine was able to significantly enhance the mucosal IgA level. The inclusion of recombinant cholera toxin B subunit as a mucosal adjuvant along with BacHA vaccine did not influence either the systemic or mucosal immunity. Interestingly, an inactivated form of BacHA was able to induce only a negligible level of immune responses compared to its live counterpart. Microneutralization assay also indicated that live BacHA vaccine was able to induce strong cross-clade neutralization against heterologous H5N1 strains (clade 1.0, clade 2.1, and clade 8.0) compared to the inactivated BacHA. Viral challenge studies showed that live BacHA was able to provide 100% protection against 5 50% mouse lethal doses (MLD₅₀) of homologous (clade 2.1) and heterologous (clade 1) H5N1. Moreover, histopathological examinations revealed that mice vaccinated with live BacHA had only minimal bronchitis in lungs and regained their body weight more rapidly postchallenge. Furthermore, immunohistochemistry results demonstrated that the live BacHA was able to transduce and express HA in the intestinal epithelial cells in vitro and in vivo. We have demonstrated that recombinant baculovirus with a white spot syndrome virus (WSSV) immediate-early promoter 1 (ie1) acted as a vector as well as a protein vaccine and will enable the rapid production of prepandemic and pandemic vaccines without any biosafety concerns.The recent outbreaks of H5N1 avian flu and the current pandemic situation with H1N1 swine-origin influenza A virus (S-OIV) are clear indications of the urgent need for effective vaccines against influenza A viruses (31). Preventive and therapeutic measures against influenza A viruses have received much interest and effort globally to combat the current pandemic and to prevent such a situation in the future. Currently used vaccines for influenza are administered mainly parenterally and include live attenuated reassortant viruses, conventional inactivated whole viral antigens, or split-virus vaccines. Although some of these vaccines have proven to be quite effective, the manufacturing of these vaccines involves several technical and safety issues (21). Furthermore, the production of currently available influenza vaccines often requires high-level biocontainment facilities, an additional hurdle that limits the advancement of present vaccines.Vaccines containing purified recombinant viral proteins have recently gained special attention due to their ease of production without any safety concerns (25). Recombinant hemagglutinin (rHA) subunit vaccines produced in baculovirus-insect cell expression systems have been extensively tested and evaluated in humans (29, 30). Baculovirus-derived rHA subunit vaccines administered parenterally are safe and immunogenic in animals and humans. Along with its success in recombinant protein vaccines, baculovirus surface display technology allows us to present large complex proteins on the baculovirus envelope in its native antigenic conformation, resulting in good stability and a longer half-life in the host (18, 14, 8).Along with a suitable antigen, the route of administration of the vaccine has a profound effect in controlling mucosally acquired infections such as influenza. Vaccination via the mucosal route stimulates both systemic and mucosal immune responses (16). Oral and intranasal vaccines are the two main options for mucosal administration. Intranasal vaccines would have a detrimental effect on persons with asthma, reactive airway disease, and other chronic pulmonary or cardiovascular disorders (4). Oral vaccines therefore seem to be the safest alternative (13). Moreover, there is evidence to prove the ability of oral vaccination to prevent infection of the lungs (23) and cause transcytosis of the molecule across the cells into the circulation (24).In this report, we describe the construction of recombinant baculovirus under control of the immediate-early promoter 1 (ie1) derived from the white spot syndrome virus (WSSV) genome, which enables the expression of hemagglutinin at the early stage of infection in insect cells, thereby enhancing the display of HA on the baculovirus envelope. Incorporation of more HA into the budding baculovirus particles would improve their efficacy as immunogens. We have studied the efficacy of WSSV ie1-based baculovirus displaying hemagglutinin (BacHA) as an oral vaccine in a mouse model of infection. We have also assessed its efficacy with recombinant cholera toxin B (rCTB) as a mucosal adjuvant. This strategy will enable rapid production of prepandemic vaccines with minimal infrastructure around the world, alleviating the need for high-biosafety facilities, risky inactivation of virulent viruses, and meticulous protein purification procedures.     

Deficiency of Autophagy in Dendritic Cells Protects against Experimental Autoimmune Encephalomyelitis

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) in the immune system. DCs present antigens to CD8 and CD4 T cells in the context of class I or II MHC. Recent evidence suggests that autophagy, a conserved intracellular degradation pathway, regulates class II antigen presentation. In vitro studies have shown that deletion of autophagy-related genes reduced antigen presentation by APCs to CD4 T cells. In vivo studies confirmed these findings in the context of infectious diseases. However, the relevance of autophagy-mediated antigen presentation in autoimmunity remains to be elucidated. Here, we report that loss of autophagy-related gene 7 (Atg7) in DCs ameliorated experimental autoimmune encephalomyelitis (EAE), a CD4 T cell-mediated mouse model of multiple sclerosis, by reducing in vivo priming of T cells. In contrast, severity of hapten-induced contact hypersensitivity, in which CD8 T cells and NK cells play major roles, was unaffected. Administration of the autophagy-lysosomal inhibitor chloroquine, before EAE onset, delayed disease progression and, when administered after the onset, reduced disease severity. Our data show that autophagy is required in DCs for induction of EAE and suggest that autophagy might be a potential target for treating CD4 T cell-mediated autoimmune conditions.     

Mucosal Immunization with Integrase-Defective Lentiviral Vectors Protects against Influenza Virus Challenge in Mice

Recent reports highlight the potential for integrase-defective lentiviral vectors (IDLV) to be developed as vaccines due to their ability to elicit cell-mediated and humoral immune responses after intramuscular administration. Differently from their integrase-competent counterpart, whose utility for vaccine development is limited by the potential for insertional mutagenesis, IDLV possess a mutation in their integrase gene that prevents genomic integration. Instead, they are maintained as episomal DNA circles that retain the ability to stably express functional proteins. Despite their favorable profile, it is unknown whether IDLV elicit immune responses after intranasal administration, a route that could be advantageous in the case of infection with a respiratory agent. Using influenza as a model, we constructed IDLV expressing the influenza virus nucleoprotein (IDLV-NP), and tested their ability to generate NP-specific immune responses and protect from challenge in vivo. We found that administration of IDLV-NP elicited NP-specific T cell and antibody responses in BALB/c mice. Importantly, IDLV-NP was protective against homologous and heterosubtypic influenza virus challenge only when given by the intranasal route. This is the first report demonstrating that IDLV can induce protective immunity after intranasal administration, and suggests that IDLV may represent a promising vaccine platform against infectious agents.     

Nanodisc-Incorporated Hemagglutinin Provides Protective Immunity against Influenza Virus Infection

Every year, influenza virus infection causes significant mortality and morbidity in human populations. Although egg-based inactivated viral vaccines are available, their effectiveness depends on the correct prediction of the circulating viral strains and is limited by the time constraint of the manufacturing process. Recombinant subunit vaccines are easier to manufacture with a relatively short lead time but are limited in their efficacy partly because the purified recombinant membrane proteins in the soluble form most likely do not retain their native membrane-bound structure. Nanodisc (ND) particles are soluble, stable, and reproducibly prepared discoid shaped nanoscale structures that contain a discrete lipid bilayer bound by two amphipathic scaffold proteins. Because ND particles permit the functional reconstitution of membrane/envelope proteins, we incorporated recombinant hemagglutinin (HA) from influenza virus strain A/New Caledonia/20/99 (H1N1) into NDs and investigated their potential to elicit an immune response to HA and confer immunity to influenza virus challenge relative to the commercial vaccines Fluzone and FluMist. HA-ND vaccination induced a robust anti-HA antibody response consisting of predominantly the immunoglobulin G1 (IgG1) subclass and a high hemagglutination inhibition titer. Intranasal immunization with HA-ND induced an anti-HA IgA response in nasal passages. HA-ND vaccination conferred protection that was comparable to that of Fluzone and FluMist against challenge with influenza virus strain A/Puerto Rico/8/1934 (H1N1).The influenza A virus-type viral genome encodes 11 proteins including hemagglutinin (HA) and neuraminidase (NA). HA is important in virus transmission and is also a major determinant of host range (16). NA prevents viral aggregation and helps in the release of new viruses from the infected cell (25). These glycoproteins are the principal antigens against which humoral immune responses of the host are directed. Vaccination has been accepted as the most effective method of preventing influenza virus. Current licensed vaccines against influenza virus include conventional inactivated virus vaccine, live-attenuated vaccine, or inactivated “split-virus” vaccines, all grown in embryonated chicken eggs. Influenza virus vaccines may contain residual egg-derived antigens, which is a risk factor for persons with hypersensitivity to eggs. In the case of live-attenuated vaccines that are delivered by the mucosal route, there are several potential safety concerns including the possibility that the vaccine strain could undergo spontaneous genetic change and in a rare case of simultaneous infection with another influenza virus could undergo antigenic shift. These factors are of special concern for children and the elderly, who are the primary populations at risk for influenza virus infection (9). Therefore, there is a continuing need for developing more efficacious and safer vaccines.Apart from licensed vaccines, a number of different vaccine formulations including soluble glycoproteins, virus-like particles, and subunit vaccines (6, 9, 14) with various efficacies have been developed. Recombinant glycoprotein vaccines offer many distinct advantages, including cost, the possibility of adapting them to rapidly changing strains within a short time, and independence from egg-based formulations. In experimental setups, recombinant HA (rHA) and recombinant NA have provided protection against lethal challenge to mice (18, 27). The safety, immunogenicity, and efficacy of trivalent rHA vaccines have been established (26), and a potential trivalent HA vaccine (FluBlok; Protein Sciences Corporation) is currently in phase III clinical trials.Some rHA-based vaccines elicit high titers of anti-HA antibodies. However, these antibodies do not necessarily possess a high capacity for virus neutralization. This apparent discrepancy likely results from the use of soluble HA protein that may not accurately mimic the native structure of the membrane-embedded glycoprotein on the viral envelope for immunization. This could result in a robust antibody response with a limited ability to react with “native epitopes.” This notion is supported by data from previously reported studies that indicated that antigens expressed in their native three-dimensional conformation can elicit a more effective antibody response than proteins in their nonnative forms (19). Therefore, we investigated whether rHA presented in a lipid-bilayer-embedded formulation would elicit a potent neutralizing antibody response.The Nanodisc (ND) system was developed as a novel method for functionally reconstituting membrane proteins into soluble nanoscale lipid bilayers (3, 4, 12, 22). NDs are robust, reproducible, and monodisperse discoidal particles 5.5 nm high and nominally 10 nm in diameter that are formed via a self-assembly process. ND particles contain two copies of an alpha-helical, amphipathic protein, termed membrane scaffold protein (MSP), which encircles a lipid bilayer in a “belt-like” fashion (Fig. ​(Fig.1a).1a). A mixture of phospholipids and MSP are placed in a nonequilibrium solubilized state, for instance, using detergent or high hydrostatic pressure, and the system is then allowed to approach equilibrium by the gentle removal of the perturbant. This initiates a process of self-assembly, wherein the phospholipids and MSP find each other and generate a discoidal phospholipid bilayer encircled by the MSP. The resulting nanostructures represent a highly stable and homogeneous population with an aqueous solubility in the millimolar range (11).Open in a separate windowFIG. 1.Construction of HA-NDs. (a) Schematic showing an ND particle that contains a phospholipid bilayer encircled by membrane scaffold proteins (left) (5) and the same ND particle with an embedded transmembrane protein (right) (17). (b) HA-ND assemblies were first purified by Ni²⁺ affinity chromatography. (Top left) Silver-stained SDS-PAGE showing flowthrough, wash, and elution of HA-ND assembly mix over a Ni-nitrilotriacetic acid column (FT1 and FT2 are flowthrough, and the eluate contains the eluted protein). Arrows show the positions of the 72-kDa HA band and the 25-kDa MSPs. (Top right) Anti-HA Western blotting of the same SDS-PAGE gel. Depending on the quality of purification, a certain fraction of full-length 72-kDa rHA (HA0) can exist as proteolytically cleaved HA1 (∼50-kDa) and HA2 (∼28-kDa) subunits. (Bottom left) Ni²⁺ column eluates were further purified by SEC. Silver-stained SDS-PAGE gel shows size-based fractionation of Ni²⁺ column eluate. The numbers at the bottom correspond to the fractions collected. The MSP amounts are largest at fractions 27 to 30, showing that empty NDs eluted at those fractions. (Bottom right) Anti-HA Western blotting of the same SDS-PAGE gel showing that HA-ND assemblies eluted mainly between fractions 18 and 26. (c) Elution profile of HA-ND following SEC separation. The elution times for protein standards used for calibration are indicated at the top. The control profile for empty NDs is superimposed. HA-ND assemblies have a shorter retention time than empty NDs. inj, injection. (d) HA-ND assemblies from different SEC fractions separate as discrete-sized molecules upon native PAGE separation. Silver staining (left) and anti-HA Western blotting (right) of native PAGE gels from size exclusion fractions show different HA polymers contained in NDs. Earlier fractions are rich in higher-polymeric forms of HA, while later fractions are richer in monomeric HA. Control HA was loaded in the last well to the right in both cases.The value of the ND self-assembly process is that one can simply and reproducibly incorporate membrane proteins into these structures. This is accomplished by including the membrane protein in the initial mixture of MSP, lipid, and detergent prior to the initiation of the self-assembly process. An incorporated membrane protein then finds itself in a native-like environment with stability and activity normally found in vivo. By using phospholipids with different chemical characteristics (charge, degree of unsaturation, and length of acyl chains), the bilayer environment can be optimized to accommodate functional requirements. Furthermore, larger scaffold proteins, which in turn create a larger-diameter particle, can be employed to incorporate multimers or membrane protein complexes. Numerous membrane proteins from the three major classes-integral, tethered, and embedded (including monomers and multimers)-in the lipid bilayer environment created by NDs have been studied (2-5, 8, 10, 13, 20, 23). Since the ND system creates a stable bilayer environment that mimics that encountered by a membrane protein in the cell membrane, membrane proteins display normal folding, native ligand binding kinetics, and intact signaling activity (1, 3, 5, 8, 10, 13, 17, 23).In this study, we successfully incorporated recombinant baculovirus-derived HA into NDs (HA-ND) and compared its efficacy to induce a relevant immune response and confer protection against influenza virus challenge with those of existing licensed vaccines by using a mouse model.     

Two DNA Aptamers against Avian Influenza H9N2 Virus Prevent Viral Infection in Cells

New antiviral therapy for pandemic influenza mediated by the H9N2 avian influenza virus (AIV) is increasingly in demand not only for the poultry industry but also for public health. Aptamers are confirmed to be promising candidates for treatment and prevention of influenza viral infections. Thus, we studied two DNA aptamers, A9 and B4, selected by capillary electrophoresis-based systemic evolution of ligands by exponential enrichment (CE-SELEX) procedure using H9N2 AIV purified haemagglutinin (HA) as target. Both aptamers had whole-virus binding affinity. Also, an enzyme-linked aptamer assay (ELAA) confirmed binding affinity and specificity against other AIV subtypes. Finally, we studied aptamer-inhibitory effects on H9N2 AIV infection in Madin–Darby canine kidney (MDCK) cells and quantified viral load in supernatant and in cell with quantitative PCR (qPCR). Our data provide a foundation for future development of innovative anti-influenza drugs.