Cheryl M. Cameron

Gene Expression Analysis of Host Innate Immune Responses during Lethal H5N1 Infection in Ferrets

ABSTRACT How viral and host factors contribute to the severe pathogenicity of the H5N1 subtype of avian influenza virus infection in humans is poorly understood. We identified three clusters of differentially expressed innate immune response genes in lungs from H5N1 (A/Vietnam/1203/04) influenza virus-infected ferrets by oligonucleotide microarray analysis. Interferon response genes were more strongly expressed in H5N1-infected ferret lungs than in lungs from ferrets infected with the less pathogenic H3N2 subtype. In particular, robust CXCL10 gene expression in H5N1-infected ferrets led us to test the pathogenic role of signaling via CXCL10s cognate receptor, CXCR3, during H5N1 influenza virus infection. Treatment of H5N1-infected ferrets with the drug AMG487, a CXCR3 antagonist, resulted in a reduction of symptom severity and delayed mortality compared to vehicle treatment. We contend that unregulated host interferon responses are at least partially responsible for the severity of H5N1 infection and provide evidence that attenuating the CXCR3 signaling pathway improves the clinical course of H5N1 infection in ferrets.

Interferon-Mediated Immunopathological Events Are Associated with Atypical Innate and Adaptive Immune Responses in Patients with Severe Acute Respiratory Syndrome

ABSTRACT It is not understood how immune inflammation influences the pathogenesis of severe acute respiratory syndrome (SARS). One area of strong controversy is the role of interferon (IFN) responses in the natural history of SARS. The fact that the majority of SARS patients recover after relatively moderate illness suggests that the prevailing notion of deficient type I IFN-mediated immunity, with hypercytokinemia driving a poor clinical course, is oversimplified. We used proteomic and genomic technology to systematically analyze host innate and adaptive immune responses of 40 clinically well-described patients with SARS during discrete phases of illness from the onset of symptoms to discharge or a fatal outcome. A novel signature of high IFN-α, IFN-γ, and IFN-stimulated chemokine levels, plus robust antiviral IFN-stimulated gene (ISG) expression, accompanied early SARS sequelae. As acute illness progressed, SARS patients entered a crisis phase linked to oxygen saturation profiles. The majority of SARS patients resolved IFN responses at crisis and expressed adaptive immune genes. In contrast, patients with poor outcomes showed deviated ISG and immunoglobulin gene expression levels, persistent chemokine levels, and deficient anti-SARS spike antibody production. We contend that unregulated IFN responses during acute-phase SARS may culminate in a malfunction of the switch from innate immunity to adaptive immunity. The potential for the use of the gene signatures we describe in this study to better assess the immunopathology and clinical management of severe viral infections, such as SARS and avian influenza (H5N1), is therefore worth careful examination.

Modeling host responses in ferrets during A/California/07/2009 influenza infection.

Immune responses during infection with pandemic H1N1 2009 influenza A virus (2009-H1N1) are still poorly understood. Using an experimental infection model in ferrets, we examined the pathological features and characterized the host immune responses by using microarray analysis, during infection with 2009-H1N1 A/California/07/2009 and seasonal A/Brisbane/59/2007. Chemokines CCL2, CCL8, CXCL7 and CXCL10 along with the majority of interferon-stimulated genes were expressed early, correlated to lung pathology, and abruptly decreased expression on day 7 following infection of A/California/07/2009. Interestingly, the drop in innate immune gene expression was replaced by a significant increase of the adaptive immune genes for granzymes and immunoglobulins. Serum anti-influenza antibodies were first observed on day 7, commensurate with the viral clearance. We propose that lung pathology in humans occurs during the innate phase of host immunity and a delay or failure to switch to the adaptive phase may contribute to morbidity and mortality during severe 2009-H1N1 infections.

Myxoma Virus M141R Expresses a Viral CD200 (vOX-2) That Is Responsible for Down-Regulation of Macrophage and T-Cell Activation In Vivo

ABSTRACT M141R is a myxoma virus gene that encodes a cell surface protein with significant amino acid similarity to the family of cellular CD200 (OX-2) proteins implicated in the regulation of myeloid lineage cell activation. The creation of an M141R deletion mutant myxoma virus strain (vMyx141KO) and its subsequent infection of European rabbits demonstrated that M141R is required for the full development of a lethal infection in vivo but is not required for efficient virus replication in susceptible cell lines in vitro. Minor secondary sites of infection were detected in the majority of rabbits infected with the M141R deletion mutant, demonstrating that the M141R protein is not required for the dissemination of virus within the host. When compared to wild-type myxoma virus-infected rabbits, vMyx141KO-infected rabbits showed higher activation levels of both monocytes/macrophages and lymphocytes in situ through assessments of inducible nitric oxide synthase-positive and CD25+ infiltrating cells in infected and lymphoid tissues. Purified peripheral blood mononuclear cells from vMyx141KO-infected rabbits demonstrated an increased ability to express gamma interferon upon activation by phorbol myristate acetate plus ionomycin compared to cells purified from wild-type myxoma virus-infected rabbits. We concluded that the M141R protein is a bona fide CD200-like immunomodulator protein which is required for the full pathogenesis of myxoma virus in the European rabbit and that its loss from the virus results in increased activation levels of macrophages in infected lesions and draining lymph nodes as well as an increased activation level of circulating T lymphocytes during infection. We propose a model whereby M141R transmits inhibitory signals to tissue macrophages, and possibly resident CD200R+ dendritic cells, that reduce their ability to antigenically prime lymphocytes and possibly provides anergic signals to T cells directly.

Influenza viremia and the potential for blood-borne transmission

I nfluenza is a major cause of morbidity and mortality in the United States and worldwide. The threat of pandemic influenza recently has gained prominent attention because of widespread infection of poultry with highly pathogenic avian influenza A (H5N1) and the potential for the virus to mutate into one capable of efficient human-to-human transmission. As of March 8, 2007, 277 human cases of H5N1 infection had been reported to WHO from Asia, Eastern Europe, and Africa, mostly as a result of close contact between humans and infected birds, although rare, unsustained human-to-human transmission has been documented. If a change in viral characteristics were to allow efficient human-to-human transmission, rapid spread and a worldwide pandemic could result. The global spread of H5N1, continuing outbreaks in birds, and sporadic infections in humans have increased concern that a pandemic virus may emerge and cause an influenza pandemic. The possibility of an influenza pandemic has focused attention on the epidemiology and pathophysiology of influenza, including its potential for transmission through the blood supply. An infectious agent which has a blood-borne phase and can be clinically asymptomatic has the potential to be transmitted by blood transfusion. Planning to ensure an adequate and safe national blood supply during a pandemic prompted consideration of the potential for transfusion-transmitted influenza. Notably, no cases of transfusion transmission of influenza have been documented to date. The risk of transfusion transmission has been assumed to be negligible based on the premise that viremia rarely occurs and does not occur without symptoms, allowing for deferral of potentially infectious blood donors. If these assumptions are incorrect, however, and influenza infection commonly involves a viremic phase, especially before the onset of symptoms, and if influenzainfected blood resulted in clinical illness, it would have implications for blood safety. To further examine this possibility, we surveyed the literature underlying these assumptions and propose methods of redressing gaps in our knowledge. If influenza were transmissible by transfusion, blood product recipients, who include a high proportion of immunocompromised patients, might suffer increased morbidity and mortality. In bone marrow transplant populations influenza infection appears to be associated with approximately 25 percent mortality. If influenza were deemed to represent a transfusion transmission risk, possible screening methods would likely rely on epidemiologic query or laboratory screening. With any screening there is a risk of impacting supply. Testing of the blood supply would also become particularly important if a pandemic were to be caused by a virus similar to the highly pathogenic influenza A (H5N1). Infection in humans has thus far resulted in approximately 60 percent case fatality ratio where the virus has been isolated outside of the respiratory tract, a factor not typically seen with seasonal influenza infections. Detection of influenza in the blood of H5N1 infected humans and in animal studies has occurred predominantly during symptomatic periods and at high viral titers. A positive test might result in discarding ABBREVIATION: CSF = cerebrospinal fluid.

The role of chemokines and chemokine receptors in alloantigen-independent and alloantigen-dependent transplantation injury.

Transplantation injury and rejection involves the interplay of innate and acquired immune responses. Immune-related injury manifests itself in three temporal phases: early innate immune driven alloantigen-independent injury, acquired immune driven alloantigen-dependent injury, and chronic injury. Sequential waves of chemokine expression play a central role in regulating graft injury through the recruitment of phagocytes shortly after transplantation and activated lymphocytes and phagocytes in the weeks and years following transplantation. This review focuses on recent studies demonstrating the role of chemokines in transplantation.

Immunogenicity of a receptor-binding domain of SARS coronavirus spike protein in mice: Implications for a subunit vaccine

Abstract We studied the immunogenicity of an anti-SARS subunit vaccine comprised of the fragment of the SARS coronavirus (SARS-CoV) spike protein amino acids 318–510 (S318–510) containing the receptor-binding domain. The S protein fragment was purified from the culture supernatant of stably transformed HEK293T cells secreting a tagged version of the protein. The vaccine was given subcutaneously to 129S6/SvEv mice in saline, with alum adjuvant or with alum plus CpG oligodeoxynucleotides (ODN). Mice immunized with the adjuvanted antigen elicited strong antibody and cellular immune responses; furthermore, adding the CpG ODN to the alum resulted in increased IgG2a antibody titers and a higher number of INF-γ-secreting murine splenocytes. Mice vaccinated with S318–510 deglycosylated by PNGase F (dgS318–510) showed a lower neutralizing antibody response but had similar numbers of INF-γ-producing cells in the spleen. This finding suggests that carbohydrate is important for the immunogenicity of the S318–510 protein fragment and provide useful information for designing an effective and safe SARS subunit vaccine.

Cloning, expression and immunoassay detection of ferret IFN-γ

Summary Ferrets (Mustela putorius furo) develop symptoms upon influenza infection that resemble those of humans, including sneezing, body temperature variation and weight loss. Highly pathogenic strains of influenza A, such as H5N1, have the capacity to cause severe illness or death in ferrets. The use of ferrets as a model of influenza infection is currently limited by a lack of species-specific immunological reagents. Interferon gamma (IFN-γ) plays a key role in the development of innate and adaptive immunity and the regulation of Th1-type immune responses. Here we describe the cloning of the full-length cDNA for ferret IFN-γ. Multiple sequence alignment of the predicted amino acid sequence with those of other species indicates that the predicted ferret protein shares the highest identity with Eurasian badger IFN-γ. We raised two hybridoma clones expressing monoclonal antibodies against recombinant ferret IFN-γ capable of detecting IFN-γ protein derived from mitogen-stimulated ferret PBMCs by immunoblotting, ELISA and ELISPOT assay. Finally, an ELISA utilizing the ferret-specific antibodies detected elevated levels of IFN-γ in serum samples from H3N2 influenza A-infected ferrets.

Early gene expression events in ferrets in response to SARS coronavirus infection versus direct interferon-alpha2b stimulation

Abstract Type I interferons (IFNs) are essential to the clearance of viral diseases, however, a clear distinction between genes upregulated by direct virus–cell interactions and genes upregulated by secondary IFN production has not been made. Here, we investigated differential gene regulation in ferrets upon subcutaneous administration of IFN-α2b and during SARS-CoV infection. In vivo experiments revealed that IFN-α2b causes STAT1 phosphorylation and upregulation of abundant IFN response genes (IRGs), chemokine receptors, and other genes that participate in phagocytosis and leukocyte transendothelial migration. During infection with SARS-CoV not only a variety of IRGs were upregulated, but also a significantly broader range of genes involved in cell migration and inflammation. This work allowed dissection of several molecular signatures present during SARS-CoV which are part of a robust IFN antiviral response. These signatures can be useful markers to evaluate the status of IFN responses during a viral infection and specific features of different viruses.

Cloning, expression and characterization of ferret CXCL10.

Abstract Chemokines and their receptors function in the recruitment and activation of cells of the immune system to sites of inflammation. As such, chemokines play an important role in mediating pathophysiological events during microbial infection. In particular, CXCL9, CXCL10 and CXCL11 and their cognate receptor CXCR3 have been associated with the clinical course of several infectious diseases, including severe acute respiratory syndrome (SARS) and influenza. While CXCL9, CXCL10 and CXCL11 share the same receptor and have overlapping functions, each can also have unique activity in host defense. The lack of a preferred characterized animal model for SARS has brought our attention to ferrets, which have been used for years in influenza studies. The lack of immunological reagents for ferrets prompted us to clone CXCL9, CXCL10, CXCL11 and CXCR3 and, in the case of CXCL10, to express the gene as a recombinant protein. In this study we demonstrate that endogenous ferret CXCL10 exhibits similar mRNA expression patterns in the lungs of deceased SARS patients and ferrets experimentally infected with SARS coronavirus. This study therefore represents an important step towards development of the ferret as a model for the role of CXCL9, CXCL10 and CXCL11:CXCR3 axis in severe viral infections.