Cellular immunity in Pneumovirus infections

Human Respiratory Syncytial Virus (RSV) is the leading cause of viral respiratory tract infection in infants worldwide. In the developed world viral bronchiolitis is the most common cause of hospitalization among infants, 70% of these are associated with RSV. In recent years the realization is growing that RSV also is a considerable disease burden on adults as well. At the moment there is no effective antiviral therapy or an effective vaccine available against RSV infection. The development of a safe and working vaccine would greatly benefit from a better understanding of the interactions between the virus and its host. RSV is a negative sense, single-stranded RNA virus, part of the family of Paramyxoviridae, subfamily Pneumovirinae, genus Pneumovirus. Two closely related viruses Bovine Repiratory Syncytial virus (BRSV) and Pneumonia Virus of Mice (PVM) cause in their hosts, cattle and mice respectively, clinical symptoms reminiscent of RSV disease in humans. The aim of the research described in this thesis was to gain better insight in the (cellular) immunological responses of the hosts against pneumoviruses. Three models were used to examine the cellular immunity against pneumoviral infection, RSV in mice, BRSV in cattle, and a relatively new model, PVM in mice. Since the murine RSV model relies heavily on BALB/c mice, we have investigated the cellular immunity against RSV in C57BL/6. Five RSV-specific CD8 T cell epitopes were identified. Using the BRSV cattle infection model, the antiviral CD8 T cell reponses are analyzed. In the last model, PVM in mice, the first antiviral CD8 and CD4 T cell responses are described and their role in protection against PVM infection is examined. The results obtained with PVM in mice argue that it could represent a promising small animal model to elucidate pneumovirus host interactions

Activation and inactivation of antiviral CD8 T cell responses during murine pneumorvirus infection

Pneumonia virus of mice (PVM) is a natural pathogen of mice and has been proposed as a tractable model for the replication of a pneumovirus in its natural host, which mimics human infection with human respiratory syncytial virus (RSV). PVM infection in mice is highly productive in terms of virus production compared with the situation seen with RSV in mice. Because RSV suppresses CD8 T cell effector function in the lungs of infected mice, we have investigated the nature of PVM-induced CD8 T cell responses to study pneumovirus-induced T cell responses in a natural virus-host setting. PVM infection was associated with a massive influx of activated CD8 T cells into the lungs. After identification of three PVM-specific CD8 T cell epitopes, pulmonary CD8 T cell responses were enumerated. The combined frequency of cytokine-secreting CD8 T cells specific for the three epitopes was much smaller than the total number of activated CD8 T cells. Furthermore, quantitation of the CD8 T cell response against one of these epitopes (residues 261¿270 from the phosphoprotein) by MHC class I pentamer staining and by in vitro stimulation followed by intracellular IFN- and TNF- staining indicated that the majority of pulmonary CD8 specific for the P261 epitope were deficient in cytokine production. This deficient phenotype was retained up to 96 days postinfection, similar to the situation in the lungs of human RSV-infected mice. The data suggest that PVM suppresses T cell effector functions in the lungs

Porcine beta-defensin 2 displays broad antimicrobial activity against pathogenic intestinal bacteria

Defensins are small antimicrobial peptides that play an important role in the innate immune system of mammals. Here, we describe the antimicrobial activity of pBD-2, a recently discovered new porcine defensin that is produced in the intestine. A synthetic peptide corresponding to the mature protein showed high antimicrobial activity against a broad range of pathogenic bacteria, while it only showed limited hemolytic activity against porcine red blood cells. Highest activity was observed against Salmonella typhimurium, Listeria monocytogenes and Erysipelothrix rhusiopathiae. pBD-2 (4¿8 ¿M) killed these pathogens within 3 h. The activity of pBD-2 against S. typhimurium was studied in more detail. At the minimum bactericidal concentration (MBC) of pBD-2, complete killing of S. typhimurium was relatively fast with no viable bacteria left after 90 min. However, antimicrobial activity of pBD-2 was decreased at higher ionic strengths with no residual activity at 150 mM NaCl. Transmission electron microscopy of pBD-2 treated S. typhimurium indicated that relatively low doses of pBD-2 caused a retraction of the cytoplasmic membrane, while pBD-2 concentrations close to the MBC led to cytoplasm leakage and complete lysis of bacterial cells. Considering the site of production and the activity, pBD-2 may be an important defense molecule for intestinal health

Growth phase of orally administered Lactobacillus strains differentially affects T helper-cell pathways

Lactobacillus strains with probiotic activity are major constituents of numerous common food products. Due to their `generally regarded as safe¿-status (GRAS-status), Lactobacillus strains can also be genetically engineered for use in oral immunotherapeutic applications, such as vaccination and T lymphocyte tolerance induction in autoimmune disease. In the current study, we demonstrate that the growth phase of orally administered individual Lactobacillus strains can differentially affect antigen-specific antibody subclasses IgG1 and IgG2a, which might reflect skewing of systemic activity of T helper cell type 2 (Th2) and T helper cell type 1 (Th1) pathways, respectively. Mice were orally fed different wild type Lactobacillus strains in log phase or stationary phase and immunized intraperitoneally with a T-cell dependent protein antigen. Sera were evaluated for the ratio of antigen-specific IgG1 and IgG2a antibodies. Stationary Lactobacillus murines and Lactobacillus casei cultures, but not two other Lactobacillus strains, evoked significantly higher IgG1/IgG2a ratios than log phase cultures, possibly relating to increased activity of the Th2-pathway. Despite normal variation in antibody responses against TNP¿CGG among individual mice, a high correlation was found between the IgG1 and IgG2a responses of mice within experimental groups. This differential antibody response is likely due to growth phase-dependent differences in bacterial cell composition. Since Lactobacillus growth phase dependent skewing of antibody responses possibly reflecting T-cell pathways can inadvertently affect allergic and (auto)-immune responses, the current findings strongly caution against unidimensional views on the oral administration of individual Lactobacillus strains for probiotic or immunotherapeutic purposes, but also suggest additional possibilities for immune modulation

Identification of aCD4 T cell epitope in the pneumonia virus of mice glycoprotein and characterization of its role in protective immunity

Pneumonia virus of mice (PVM) causes bronchiolitis and pneumonia in mice. Infection is associated with high levels of viral replication in the lungs and results in the functional inactivation of pulmonary virus-specific CD8 T cells. Due to its similarity to severe human respiratory syncytial virus (RSV) infection, PVM infection in mice has been proposed as an alternative RSV model. Here, we have delineated the minimal requirements for protective T cell immunity in the PVM model. Immunization with a CD8 T cell epitope from the PVM phosphoprotein P, combined with the ovalbumin (OVA) CD4 T cell epitope, did not confer protective immunity against lethal PVM challenge, suggesting a possible role of cognate CD4 T cell immunity. To determine the role of PVM-specific CD4 T cell responses, we mapped a PVM CD4 T cell epitope in the glycoprotein G, using a panel of overlapping peptides. Although immunization with this epitope provided some protection, solid protective immunity was only observed after immunization with a combination of the PVM-specific CD4 and CD8 T cell epitopes. Analysis of post-challenge T cell responses in immunized mice indicated that G-specific pulmonary CD4 T cells displayed a mixed Th1/Th2 phenotype, which was characterized by the presence of both IL-5 and IFN-gamma secreting cells, in the absence of overt pathology

Chicken dendritic cells are susceptible to highly pathogenic avian influenza viruses which induce strong cytokine responses

Infection with highly pathogenic avian influenza (HPAI) in birds and mammals is associated with severe pathology and increased mortality. We hypothesize that in contrast to low pathogenicity avian influenza (LPAI) infection, HPAI infection of chicken dendritic cells (DC) induces a cytokine deregulation which may contribute to their highly pathogenic nature. Infection of DC with LPAI H7N1 and H5N2 resulted in viral RNA and NP expression without increase in time, in contrast to HPAI H7N1 and H5N2 mRNA expression. No increase in IFN mRNA was detected after infection with LPAI, but after LPAI H5N2, and not LPAI H7N1, infection the level of bioactive IFNa/ß significantly increased. After HPAI H7N1 and H5N2 infection, significant increases in IL-8, IFN-a, IFN-¿ mRNA expression and in TLR1, 3, and 21 mRNA were observed. This enhanced activation of DC after HPAI infection may trigger deregulation of the immune response as seen during HPAI infection in chickens

Chicken dendritic cells are susceptible to highly pathogenic avian influenza viruses which induce strong cytokine responses

Infection with highly pathogenic avian influenza (HPAI) in birds and mammals is associated with severe pathology and increased mortality. We hypothesize that in contrast to low pathogenicity avian influenza (LPAI) infection, HPAI infection of chicken dendritic cells (DC) induces a cytokine deregulation which may contribute to their highly pathogenic nature. Infection of DC with LPAI H7N1 and H5N2 resulted in viral RNA and NP expression without increase in time, in contrast to HPAI H7N1 and H5N2 mRNA expression. No increase in IFN mRNA was detected after infection with LPAI, but after LPAI H5N2, and not LPAI H7N1, infection the level of bioactive IFNa/ß significantly increased. After HPAI H7N1 and H5N2 infection, significant increases in IL-8, IFN-a, IFN-¿ mRNA expression and in TLR1, 3, and 21 mRNA were observed. This enhanced activation of DC after HPAI infection may trigger deregulation of the immune response as seen during HPAI infection in chickens

Identification of aCD4 T cell epitope in the pneumonia virus of mice glycoprotein and characterization of its role in protective immunity

Pneumonia virus of mice (PVM) causes bronchiolitis and pneumonia in mice. Infection is associated with high levels of viral replication in the lungs and results in the functional inactivation of pulmonary virus-specific CD8 T cells. Due to its similarity to severe human respiratory syncytial virus (RSV) infection, PVM infection in mice has been proposed as an alternative RSV model. Here, we have delineated the minimal requirements for protective T cell immunity in the PVM model. Immunization with a CD8 T cell epitope from the PVM phosphoprotein P, combined with the ovalbumin (OVA) CD4 T cell epitope, did not confer protective immunity against lethal PVM challenge, suggesting a possible role of cognate CD4 T cell immunity. To determine the role of PVM-specific CD4 T cell responses, we mapped a PVM CD4 T cell epitope in the glycoprotein G, using a panel of overlapping peptides. Although immunization with this epitope provided some protection, solid protective immunity was only observed after immunization with a combination of the PVM-specific CD4 and CD8 T cell epitopes. Analysis of post-challenge T cell responses in immunized mice indicated that G-specific pulmonary CD4 T cells displayed a mixed Th1/Th2 phenotype, which was characterized by the presence of both IL-5 and IFN-gamma secreting cells, in the absence of overt pathology

Identification of a CD4 T cell epitope in the pneumonia virus of mice glycoprotein and characterization of its role in protective immunity.

Pneumonia virus of mice (PVM) causes bronchiolitis and pneumonia in mice. Infection is associated with high levels of viral replication in the lungs and results in the functional inactivation of pulmonary virus-specific CD8 T cells. Due to its similarity to severe human respiratory syncytial virus (RSV) infection, PVM infection in mice has been proposed as an alternative RSV model. Here, we have delineated the minimal requirements for protective T cell immunity in the PVM model. Immunization with a CD8 T cell epitope from the PVM phosphoprotein P, combined with the ovalbumin (OVA) CD4 T cell epitope, did not confer protective immunity against lethal PVM challenge, suggesting a possible role of cognate CD4 T cell immunity. To determine the role of PVM-specific CD4 T cell responses, we mapped a PVM CD4 T cell epitope in the glycoprotein G, using a panel of overlapping peptides. Although immunization with this epitope provided some protection, solid protective immunity was only observed after immunization with a combination of the PVM-specific CD4 and CD8 T cell epitopes. Analysis of post-challenge T cell responses in immunized mice indicated that G-specific pulmonary CD4 T cells displayed a mixed Th1/Th2 phenotype, which was characterized by the presence of both IL-5 and IFN-γ secreting cells, in the absence of overt pathology

Characterization of the CD8+ T cell responses directed against respiratory syncytial virus during primary and secondary infection in C57BL/6 mice.

The BALB/c mouse model for human respiratory syncytial virus infection has contributed significantly to our understanding of the relative role for CD4+ and CD8+ T cells to immune protection and pathogenic immune responses. To enable comparison of RSV-specific T cell responses in different mouse strains and allow dissection of immune mechanisms by using transgenic and knockout mice that are mostly available on a C57BL/ 6 background, we characterized the specificity, level and functional capabilities of CD8+ T cells during primary and secondary responses in lung parenchyma, airways and spleens of C57BL/6 mice. During the primary response, epitopes were recognized originating from the matrix, fusion, nucleo- and attachment proteins, whereas the secondary response focused predominantly on the matrix epitope. C57BL/6 mice are less permissive for hRSV infection than BALB/c mice, yet we found CD8+ T cell responses in the lungs and bronchoalveolar lavage, comparable to the responses described for BALB/c mice. © 2006 Elsevier Inc. All rights reserved. Keywords: Respiratory syncytial virus; C57BL/6; CD8+ T cells; Inactivation; Tetrame