ovalbumin and Orthomyxoviridae-Infections

Selenium status has been reported to affect immune function across many different species. Yet few studies have focused on the effect of Se status on the equine immune system. This study examined the effect of Se supplementation on vaccination response and immune function in mature horses. Twenty-eight horses were blocked by age and sex and were randomly allocated to 1 of 4 dietary treatment groups: low Se (LS), adequate Se (AS), Se-yeast (SP), and sodium selenite (SS). For 35 wk, horses allocated to LS, SP, and SS received a low-Se diet (0.06 mg/kg DM) with the intention to lower Se stores, whereas AS received an adequate Se diet (0.12 mg/kg DM). A 29-wk repletion phase was as follows: LS and AS were kept on the diets fed during the depletion period, whereas SP and SS received the depletion diet plus their respective Se supplements to achieve a dietary Se concentration of 0.3 mg/kg DM. The Se status of the horses was monitored using whole blood Se and glutathione peroxidase (GSH-Px) activity as indicators. At wk 22 and 25 of the repletion phase, horses were vaccinated intramuscularly with 10 mg ovalbumin (OVA). Horses were also vaccinated against equine influenza at wk 25. Blood samples were collected for 7 wk after initial vaccination for serum separation and at 0, 3, and 5 wk postvaccination for peripheral blood mononuclear cell (PBMC) isolation and whole blood cytokine mRNA evaluation. At wk 22 of the repletion phase, both Se and GSH-Px were greater for SP and SS compared with AS and LS (P < 0.001). Serum vitamin E was similar between treatments. Response to OVA vaccination, evaluated as OVA-specific IgG production, cytokine mRNA expression of PBMC stimulated with OVA in vitro, and lymphocyte proliferation, was unaffected by Se status. Similarly, memory response to the influenza vaccine was not affected by Se status. However, decreased mRNA expression of selected cytokines was observed in PBMC stimulated with phorbol 12-myristate 13-acetate for LS compared with AS, SP, and SS (P < 0.05). Whole blood mRNA expression of IL-10 was greater for SS compared with LS, AS, and SP (P = 0.043). Although the OVA and influenza vaccination responses were unaffected by Se status, other measures of immune function did indicate that low Se status could adversely affect cell-mediated immunity.

Topics: Animals; Dietary Supplements; Female; Horse Diseases; Horses; Immunoglobulin G; Male; Orthomyxoviridae Infections; Ovalbumin; Selenium; Sodium Selenite; Vaccination; Vitamin E

In this study we assessed the effects of antigen exposure in mice pre-sensitized with allergen following viral infection on changes in lung function, cellular responses and tight junction expression. Female BALB/c mice were sensitized to ovalbumin and infected with influenza A before receiving a second ovalbumin sensitization and challenge with saline, ovalbumin (OVA) or house dust mite (HDM). Fifteen days post-infection, bronchoalveolar inflammation, serum antibodies, responsiveness to methacholine and barrier integrity were assessed. There was no effect of infection alone on bronchoalveolar lavage cellular inflammation 15 days post-infection; however, OVA or HDM challenge resulted in increased bronchoalveolar inflammation dominated by eosinophils/neutrophils or neutrophils, respectively. Previously infected mice had higher serum OVA-specific IgE compared with uninfected mice. Mice previously infected, sensitized and challenged with OVA were most responsive to methacholine with respect to airway resistance, while HDM challenge caused significant increases in both tissue damping and tissue elastance regardless of previous infection status. Previous influenza infection was associated with decreased claudin-1 expression in all groups and decreased occludin expression in OVA or HDM-challenged mice. This study demonstrates the importance of the respiratory epithelium in pre-sensitized individuals, where influenza-infection-induced barrier disruption resulted in increased systemic OVA sensitization and downstream effects on lung function.

Topics: Airway Resistance; Animals; Bronchial Hyperreactivity; Claudin-1; Down-Regulation; Female; Influenza A virus; Methacholine Chloride; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Ovalbumin; Pyroglyphidae; Treatment Outcome

Follicular helper T (T

Topics: Animals; B-Lymphocytes; Cell Differentiation; Germinal Center; Glycolysis; Hypoxia-Inducible Factor 1, alpha Subunit; Influenza A Virus, H1N1 Subtype; Mice; Orthomyxoviridae Infections; Ovalbumin; Oxidative Phosphorylation; Signal Transduction; T-Lymphocytes, Helper-Inducer

The hygiene hypothesis is the leading concept to explain the current asthma epidemic, which is built on the observation that a lack of bacterial contact early in life induces allergic T. Because little is known about the contribution of respiratory tract viruses in this context, we evaluated the effect of prior influenza infection on the development of allergic asthma.. Mice were infected with influenza and, once recovered, subjected to an ovalbumin- or house dust mite-induced experimental asthma protocol. Influenza-polarized effector memory T (Tem) cells were transferred adoptively to allergen-sensitized animals before allergen challenge. A comprehensive in silico analysis assessed homologies between virus- and allergen-derived proteins. Influenza-polarized Tem cells were stimulated ex vivo with candidate peptides. Mice were immunized with a pool of virus-derived T-cell epitopes.. In 2 murine models we found a long-lasting preventive effect against experimental asthma features. Protection could be attributed about equally to CD4. For the first time, our results illustrate heterologous immunity of virus-infected animals toward allergens. This finding extends the original hygiene hypothesis.

Topics: Allergens; Animals; Asthma; Epitopes, T-Lymphocyte; Female; Influenza A Virus, H1N1 Subtype; Mice, Inbred BALB C; Orthomyxoviridae Infections; Ovalbumin; Peptides; Pyroglyphidae; T-Lymphocytes

Antigen-specific immunoglobulin (Ig) A plays a major role in host defense against infections in gut mucosal tissue. Follicular helper T (Tfh) cells are located in germinal centers and promote IgA production via interactions with germinal center B cells. Several studies have demonstrated that some lactic acid bacteria (LAB) strains activate the host's acquired immune system, inducing IgA secretion in the intestine. However, the precise molecular mechanisms underlying the effects of LAB on IgA production and Tfh cells are not fully resolved. Lactobacillus paracasei MCC1849 is a probiotic strain isolated from the intestine of a healthy adult. In this study, we investigated the effects of orally administered heat-killed MCC1849 on IgA production in the intestine and on Tfh cell induction in vivo. We found that orally administered MCC1849 induced antigen-specific IgA production in the small intestine, serum and lungs. We also observed that MCC1849 increased the proportion of IgA+ B cells and Tfh cells in Peyer's patches (PPs). In addition, MCC1849 increased the gene expression of IL-12p40, IL-10, IL-21, STAT4 and Bcl-6 associated with Tfh cell differentiation. These results suggest that orally administered MCC1849 enhances antigen-specific IgA production and likely affects Tfh cell differentiation in PPs.

Topics: Administration, Oral; Animals; B-Lymphocytes; Cell Differentiation; Cytokines; Hot Temperature; Immunoglobulin A, Secretory; Influenza A Virus, H1N1 Subtype; Intestine, Small; Lacticaseibacillus paracasei; Lung; Male; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Ovalbumin; Peyer's Patches; Probiotics; Proto-Oncogene Proteins c-bcl-6; T-Lymphocytes, Helper-Inducer

In contrast to responses against infectious challenge, T cell responses induced via adjuvanted subunit vaccination are dependent on interleukin-27 (IL-27). We show that subunit vaccine-elicited cellular responses are also dependent on IL-15, again in contrast to the infectious response. Early expression of interferon regulatory factor 4 (IRF4) was compromised in either IL-27- or IL-15-deficient environments after vaccination but not infection. Because IRF4 facilitates metabolic support of proliferating cells via aerobic glycolysis, we expected this form of metabolic activity to be reduced in the absence of IL-27 or IL-15 signaling after vaccination. Instead, metabolic flux analysis indicated that vaccine-elicited T cells used only mitochondrial function to support their clonal expansion. Loss of IL-27 or IL-15 signaling during vaccination resulted in a reduction in mitochondrial function, with no corresponding increase in aerobic glycolysis. Consistent with these observations, the T cell response to vaccination was unaffected by in vivo treatment with the glycolytic inhibitor 2-deoxyglucose, whereas the response to viral challenge was markedly lowered. Collectively, our data identify IL-27 and IL-15 as critical to vaccine-elicited T cell responses because of their capacity to fuel clonal expansion through a mitochondrial metabolic program previously thought only capable of supporting quiescent naïve and memory T cells.

Topics: Adjuvants, Immunologic; Aerobiosis; Allergens; Animals; Female; Glycolysis; Interleukin-15; Interleukins; Mice, Inbred C57BL; Mice, Transgenic; Orthomyxoviridae Infections; Ovalbumin; T-Lymphocytes; Vaccines, Subunit; Vaccinia

The most promising strategy to sustainably prevent infectious diseases is vaccination. However, emerging as well as re-emerging diseases still constitute a considerable threat. Furthermore, lack of compliance and logistic constrains often result in the failure of vaccination campaigns. To overcome these hurdles, novel vaccination strategies need to be developed, which fulfill maximal safety requirements, show maximal efficiency and are easy to administer. Mucosal vaccines constitute promising non-invasive approaches able to match these demands. Here we demonstrate that nanoparticle (polyphosphazenes)-based vaccine formulations including c-di-AMP as adjuvant, cationic innate defense regulator peptides (IDR) and ovalbumin (OVA) as model antigen were able to stimulate strong humoral and cellular immune responses, which conferred protection against the OVA expressing influenza strain A/WSN/OVA

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Dinucleoside Phosphates; Female; Humans; Immunity, Cellular; Immunity, Humoral; Influenza A Virus, H1N1 Subtype; Influenza Vaccines; Influenza, Human; Mice; Mice, Inbred BALB C; Nanoparticles; Organophosphorus Compounds; Orthomyxoviridae Infections; Ovalbumin; Polymers; Vaccination

Previous studies have shown that some respiratory virus infections leave local populations of tissue TRM cells in the lungs which disappear as heterosubtypic immunity declines. The location of these TRM cells and their contribution to the protective CTL response have not been clearly defined. Here, fluorescence microscopy is used to show that some CD103(+) TRM cells remain embedded in the walls of the large airways long after pulmonary immunization but are absent from systemically primed mice. Viral clearance from the lungs of the locally immunized mice precedes the development of a robust Teff response in the lungs. Whereas large numbers of virus-specific CTLs collect around the bronchial tree during viral clearance, there is little involvement of the remaining lung tissue. Much larger numbers of TEM cells enter the lungs of the systemically immunized animals but do not prevent extensive viral replication or damage to the alveoli. Together, these experiments show that virus-specific antibodies and TRM cells are both required for optimal heterosubtypic immunity, whereas circulating memory CD8 T cells do not substantially alter the course of disease.

Topics: Animals; Antibodies, Viral; Antigens, CD; CD8-Positive T-Lymphocytes; Cell Proliferation; Cross Protection; Dogs; Immunization; Immunologic Memory; Influenza A virus; Integrin alpha Chains; Lung; Lymphocyte Activation; Madin Darby Canine Kidney Cells; Mice; Orthomyxoviridae Infections; Ovalbumin; Programmed Cell Death 1 Receptor; Species Specificity; Time Factors

Vaccines that elicit robust CD8⁺ T cell responses are desirable for protection against infectious diseases and cancers. However, most vaccine adjuvants fail to elicit robust CD8⁺ T cell responses without inflammation and associated toxicity. We recently reported that self-assembling peptides that form nanofibers in physiological buffers elicited strong adjuvant-free and antigen-specific antibody responses in mice. However, whether or not such nanofibers likewise can elicit strong CD8⁺ T cell responses is unknown. Here, we demonstrate that the self-assembling peptide Q11 conjugated to a CD8⁺ T cell epitope of ovalbumin (Q11-OVA), elicits strong antigen-specific primary and recall responses, and in a vaccination regimen protects against subsequent infection. Importantly, we show that these antigenic peptide nanofibers do not persist as an inflammatory antigen depot at the injection site. Our results demonstrate for the first time that self-assembling peptides may be useful as carriers for vaccines where CD8⁺ T cell-mediated protection is needed.

Topics: Adjuvants, Immunologic; Animals; CD8-Positive T-Lymphocytes; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nanofibers; Orthomyxoviridae Infections; Ovalbumin; Peptides

Current vaccines for influenza do not fully protect the aged against influenza infection. Although wolfberry (goji berry) has been shown to improve immune response, including enhanced antibody production, after vaccination in the aged, it is not known if this effect would translate to better protection after influenza infection, nor is its underlying mechanism well understood. To address these issues, we conducted a study using a 2 × 2 design in which aged male mice (20-22 mo) were fed a control or a 5% wolfberry diet for 30 d, then immunized with an influenza vaccine or saline (control) on days 31 and 52 of the dietary intervention, and finally challenged with influenza A/Puerto Rico/8/34 virus. Mice fed wolfberry had higher influenza antibody titers and improved symptoms (less postinfection weight loss) compared with the mice treated by vaccine alone. Furthermore, an in vitro mechanistic study showed that wolfberry supplementation enhanced maturation and activity of antigen-presenting dendritic cells (DCs) in aged mice, as indicated by phenotypic change in expression of DC activation markers major histocompatibility complex class II, cluster of differentiation (CD) 40, CD80, and CD86, and functional change in DC production of cytokines interleukin-12 and tumor necrosis factor-α as well as DC endocytosis. Also, adoptive transfer of wolfberry-treated bone marrow DCs (loaded with ovalbumin(323-339)-peptide) promoted antigen-specific T cell proliferation as well as interleukin-4 and interferon-γ production in CD4(+) T cells. In summary, our data indicate that dietary wolfberry enhances the efficacy of influenza vaccination, resulting in better host protection to prevent subsequent influenza infection; this effect may be partly attributed to improved DC function.

Topics: Adjuvants, Immunologic; Animals; Antibodies; B7-1 Antigen; B7-2 Antigen; Bone Marrow; CD4-Positive T-Lymphocytes; CD40 Antigens; Cytokines; Dendritic Cells; Dietary Supplements; Endocytosis; Fruit; Genes, MHC Class II; Immunization; Influenza A virus; Influenza Vaccines; Lycium; Male; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Ovalbumin; Peptide Fragments; Phytotherapy; Plant Preparations; Weight Loss

Although morbidity and mortality rates from asthma are highest in patients > 65 years of age, the effect of older age on airway inflammation in asthma is not well established.. To investigate age-related differences in the promotion of allergic inflammation after influenza A viral respiratory infection on antigen-specific IgE production, antigen-induced airway inflammation and airway hyperresponsiveness in mice.. To accomplish this objective, the following model system was used. Young (6 week) and aged (18 months) BALB/c mice were first infected with a non-lethal dose of influenza virus A (H/HKx31). Mice were then ovalbumin (OVA)-sensitized during the acute infection (3-days post inoculation) and then chronically underwent challenge to the airways with OVA. Forty-eight hours after the final OVA challenge, airway hyperresponsiveness (AHR), bronchoalveolar fluid (BALF) cellular and cytokine profile, antigen-specific IgE and IgG1, and lung tissue inflammation were measured.. Age-specific differences were noted on the effect of a viral infection, allergic sensitization, airway inflammation and airway hyperresponsiveness. Serum OVA-specific IgE was significantly increased in only the aged mice infected with influenza virus. Despite greater morbidity (e.g. weight loss and sickness scores) during the acute infection in the 18-month old mice that were OVA-sensitized, there was little effect on the AHR and BALF cellular differential. In contrast, BALF neutrophils and AHR increased, but eosinophils decreased in 6-week mice that were OVA-sensitized during an acute influenza infection.. With increased age in a mouse model, viral infection prior to antigen sensitization affects the airway and systemic allergic response differently. These differences may reflect distinct phenotypic features of allergic inflammation in older patients with asthma.

Topics: Age Factors; Animals; Antigens; Asthma; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Female; Immunoglobulin E; Influenza A virus; Leukocyte Count; Lung; Mice; Orthomyxoviridae Infections; Ovalbumin; Respiratory Hypersensitivity

The addition of an appropriate adjuvant that activates the innate immunity is essential to subsequent development of the adaptive immunity specific to the vaccine antigens. Thus, any innovation capable of improving the immune responses may lead to a more efficacious vaccine. We recently identified a novel immune modulator using a naturally occurring seed peptide called lunasin. Lunasin was originally isolated from soybeans, and it is a small peptide containing 43 amino acids. Our studies revealed stimulatory effects of lunasin on innate immune cells by regulating expression of a number of genes that are important for immune responses. The objective was to define the effectiveness of lunasin as an adjuvant that enhances immune responses. The immune modulating functions of lunasin were characterized in dendritic cells (DCs) from human peripheral blood mononuclear cells (PBMCs). Lunasin-treated conventional DCs (cDCs) not only expressed elevated levels of co-stimulatory molecules (CD86, CD40) but also exhibited up-regulation of cytokines (IL1B, IL6) and chemokines (CCL3, CCL4). Lunasin-treated cDCs induced higher proliferation of allogeneic CD4+ T cells when comparing with medium control treatment in the mixed leukocyte reaction (MLR). Immunization of mice with ovalbumin (OVA) and lunasin inhibited the growth of OVA-expressing A20 B-lymphomas, which was correlated with OVA-specific CD8+ T cells. In addition, lunasin was an effective adjuvant for immunization with OVA, which together improved animal survival against lethal challenge with influenza virus expressing the MHC class I OVA peptide SIINFEKL (PR8-OTI). These results suggest that lunasin may function as a vaccine adjuvant by promoting DC maturation, which in turn enhances the development of protective immune responses to the vaccine antigens.

Topics: Adjuvants, Immunologic; Animals; Antigens, CD; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Chemokines; Cytokines; Dendritic Cells; Female; Humans; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Lymphoma, B-Cell; Mice, Inbred BALB C; Orthomyxoviridae Infections; Ovalbumin; Soybean Proteins

NOD2 is an intracellular pattern recognition receptor that provides innate sensing of bacterial muramyl dipeptide by host cells, such as dendritic cells, macrophages and epithelial cells. While NOD2's role as an innate pathogen sensor is well established, NOD2 is also expressed at low levels in T cells and there are conflicting data as to whether NOD2 plays an intrinsic role in T cell function. Here we show that following adoptive transfer into WT hosts, NOD2(-/-) OT-I T cells show a small decrease in the number of OVA-specific CD8 T cells recovered at the peak of the response to respiratory influenza virus infection. On the other hand, no such defect was observed upon intranasal immunization with a replication defective adenovirus carrying the OVA epitope recognized by OT-I, or when OVA was delivered with LPS subcutaneously, or when influenza-OVA was delivered intraperitoneally. Thus we observed a selective defect in NOD2-deficient T cell responses only during a live viral infection. Moreover, there was no apparent defect when NOD2(-/-) OT-I T cells were stimulated in vitro. Finally, this selective defect in recovery of NOD2-deficient CD8 T cells was not observed in a non-transgenic respiratory infection model in which mixed bone marrow chimeras were used such that the NOD2(-/-) T cells were allowed to develop and respond in a NOD2-sufficient host. Taken together our data indicate that T cell intrinsic NOD2 is not required for CD8 T cell responses to antigen delivered under a variety of conditions in vitro and in vivo. However, CD8 T cells that have developed in the absence of NOD2 show a selective and modest impairment in their response to live respiratory influenza infection.

Topics: Adoptive Transfer; Animals; CD8-Positive T-Lymphocytes; Flow Cytometry; Immunity, Innate; Immunization; Influenza A virus; Lipopolysaccharides; Madin Darby Canine Kidney Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nod2 Signaling Adaptor Protein; Orthomyxoviridae Infections; Ovalbumin; Receptors, Antigen, T-Cell, alpha-beta; Spleen

Individuals infected with mycobacteria are likely to experience episodes of concurrent infections with unrelated respiratory pathogens, including the seasonal or pandemic circulating influenza A virus strains. We analyzed the impact of influenza A virus and mycobacterial respiratory coinfection on the development of CD8 T cell responses to each pathogen. Coinfected mice exhibited reduced frequency and numbers of CD8 T cells specific to Mycobacterium bovis bacille Calmette-Guérin (BCG) in the lungs, and the IFN-γ CD8 T cell response to BCG-encoded OVA was decreased in the lungs of coinfected mice, when compared with mice infected with BCG alone. Moreover, after 2 wk of infection, mice coinfected with both pathogens showed a significant increase in the number of mycobacteria present in the lung compared with mice infected with BCG only. Following adoptive transfer into coinfected mice, transgenic CD8 T cells specific for OVA(257-264) failed to proliferate as extensively in the mediastinal lymph nodes as in mice infected only with BCG-OVA. Also noted was a reduction in the proliferation of BCG-specific CD4 transgenic T cells in mice coinfected with influenza compared with mice infected with BCG alone. Furthermore, phenotypic analysis of CD11c(+) dendritic cells from mediastinal lymph nodes of the infected mice showed that coinfection was associated with decreased surface expression of MHC class II and class I. Thus, concurrent pulmonary infection with influenza A virus is associated with decreased MHC expression on dendritic cells, reduced activation of BCG-specific CD4 and CD8 T cells, and impaired clearance of mycobacteria.

Topics: Animals; Cells, Cultured; Coculture Techniques; Female; HEK293 Cells; Humans; Influenza A virus; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mycobacterium bovis; Orthomyxoviridae Infections; Ovalbumin; T-Lymphocyte Subsets; Tuberculosis, Pulmonary

Allergic asthma is a globally respiratory inflammatory disease. Influenza virus is a respiratory pathogen that causes yearly epidemics and results in high rates of morbidity and mortality. Patients with allergic asthma had a more severe symptom and a higher mortality when they were infected with influenza virus. Hence, influenza vaccination is recommended for patients with asthma.. We evaluated the efficacy and effects of influenza vaccination on allergic asthma in a mouse model.. Ovalbumin-immunized mice were inoculated with inactivated influenza virus A/Puerto Rico/8/34 (PR8) as vaccines and morbidity or mortality and allergic asthma features of these mice were analyzed.. Mice inoculated with inactivated PR8 induced high levels of anti-PR8 IgG2a and upregulation of Toll-like receptor (TLR) 7. Vaccinated allergic mice were healthy when they were challenged with live influenza virus while none of non-vaccinated allergic mice survived. Furthermore, inactivated influenza virus vaccine induced neither extra airway inflammation nor asthma features such as IgE, airway hyper-reactivity, and eosinophilia in allergic mice. Particularly, decreased frequency of immune cell infiltrated airways and Th2 cytokines IL-4 and IL-6 production in the bronchoalveolar lavage fluid were noted in vaccinated allergic mice. These results suggested that inactivated influenza virus vaccine is efficient to protect allergic mice from further influenza infection, and it does not exacerbate but reduces IL-4 and IL-6 of allergic asthma.. Influenza vaccination is essential and efficient for allergic subjects to protect influenza virus infection.

Topics: Animals; Asthma; Disease Models, Animal; Female; Humans; Influenza Vaccines; Interleukin-4; Interleukin-6; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Ovalbumin; Puerto Rico; Vaccines, Inactivated

Different diseases require different immune responses for efficient protection. Thus, prophylactic vaccines should prime the immune system for the particular type of response needed for protection against a given infectious agent. We have here tested fusion DNA vaccines which encode proteins that bivalently target influenza hemagglutinins (HA) to different surface molecules on antigen presenting cells (APC). We demonstrate that targeting to MHC class II molecules predominantly induced an antibody/Th2 response, whereas targeting to CCR1/3/5 predominantly induced a CD8(+)/Th1 T cell response. With respect to antibodies, the polarizing effect was even more pronounced upon intramuscular (i.m) delivery as compared to intradermal (i.d.) vaccination. Despite these differences in induced immune responses, both vaccines protected against a viral challenge with influenza H1N1. Substitution of HA with ovalbumin (OVA) demonstrated that polarization of immune responses, as a consequence of APC targeting specificity, could be extended to other antigens. Taken together, the results demonstrate that vaccination can be tailor-made to induce a particular phenotype of adaptive immune responses by specifically targeting different surface molecules on APCs.

Topics: Adaptive Immunity; Animals; Antibodies, Viral; Antigen Presentation; Dendritic Cells; Female; Gene Expression; Hemagglutinin Glycoproteins, Influenza Virus; Histocompatibility Antigens Class II; Influenza A Virus, H1N1 Subtype; Influenza Vaccines; Injections, Intradermal; Injections, Intramuscular; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Ovalbumin; Receptors, CCR; Th1 Cells; Th2 Cells; Vaccination; Vaccines, Synthetic

As the main mucosal immune inductive site of nasal cavity, nasal-associated lymphoid tissue (NALT) plays an important role in both antigen recognition and immune activation after intranasal immunization. However, the efficiency of intranasal vaccines is commonly restricted by the insufficient intake of antigen by the nasal mucosa, resulting from the nasal mucosal barrier and the nasal mucociliary clearance. The distribution of NALT and the characteristic of nasal cavity have already been described in humans and many laboratory rodents, while data about poultry are scarce. For this purpose, histological sections of the chicken nasal cavities were used to examine the anatomical structure and histological characteristics of nasal cavity. Besides, the absorptive capacity of chicken nasal mucosa was also studied using the materials with different particle size. Results showed that the NALT of chicken was located on the bottom of nasal septum and both sides of choanal cleft, which mainly consisted of second lymphoid follicle. A large number of lymphocytes were distributed under the mucosal epithelium of inferior nasal meatus. In addition, there were also diffuse lymphoid tissues located under the epithelium of the concha nasalis media and the walls of nasal cavity. The results of absorption experiment showed that the chicken nasal mucosa was capable to absorb trypan blue, OVA, and fluorescent latex particles. Inactivated avian influenza virus (IAIV) could be taken up by chicken nasal mucosa except for the stratified squamous epithelium sites located on the forepart of nasal cavity. The intake of IAIV by NALT was greater than that of the nasal mucosa covering on non-lymphoid tissue, which could be further enhanced after intranasal inoculation combined with sodium cholate or CpG DNA. The study on NALT and nasal absorptive capacity will be benefit for further understanding of immune mechanisms after nasal vaccination and development of nasal vaccines for poultry.

Topics: Absorption; Administration, Intranasal; Animals; Chickens; Coloring Agents; Immunoenzyme Techniques; Influenza A virus; Lymphoid Tissue; Nasal Cavity; Nasal Mucosa; Oligodeoxyribonucleotides; Orthomyxoviridae Infections; Ovalbumin; Sodium Cholate; Trypan Blue; Vaccination; Vaccines

Infection with influenza A virus represents a major public health threat worldwide, particularly in patients with asthma. However, immunity induced by influenza A virus may have beneficial effects, particularly in young children, that might protect against the later development of asthma, as suggested by the hygiene hypothesis. Herein, we show that infection of suckling mice with influenza A virus protected the mice as adults against allergen-induced airway hyperreactivity (AHR), a cardinal feature of asthma. The protective effect was associated with the preferential expansion of CD4-CD8-, but not CD4+, NKT cells and required T-bet and TLR7. Adoptive transfer of this cell population into allergen-sensitized adult mice suppressed the development of allergen-induced AHR, an effect associated with expansion of the allergen-specific forkhead box p3+ (Foxp3+) Treg cell population. Influenza-induced protection was mimicked by treating suckling mice with a glycolipid derived from Helicobacter pylori (a bacterium associated with protection against asthma) that activated NKT cells in a CD1d-restricted fashion. These findings suggest what we believe to be a novel pathway that can regulate AHR, and a new therapeutic strategy (treatment with glycolipid activators of this NKT cell population) for asthma.

Topics: Adoptive Transfer; Animals; Animals, Suckling; Asthma; Disease Models, Animal; Forkhead Transcription Factors; Glycolipids; Helicobacter pylori; Humans; Influenza A virus; Influenza, Human; Lung; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Knockout; Models, Immunological; Natural Killer T-Cells; Orthomyxoviridae Infections; Ovalbumin; Respiratory Hypersensitivity; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory

Vaccines can greatly reduce the spread of and deaths from many infectious diseases. However, many infections have no successful vaccines. Better understanding of the generation of protective CD8 memory T cells by vaccination is essential for the rational design of new vaccines that aim to prime cellular immune responses. Here we demonstrate that the combination of two adjuvants that are currently licensed for use in humans can be used to prime long-lived memory CD8 T cells that protect mice from viral challenge. The universally used adjuvant, aluminum salts, primed long-lived memory CD8 T cells; however, effective cytotoxic T-cell differentiation occurred only in the presence of an additional adjuvant, monophosphoryl lipid A (MPL). MPL-induced IL-6 was required for cytotoxic differentiation. The IL-6 acted by inducing granzyme B production and reducing expression of inhibitory molecule PD1 on the surface of the primed CD8 T cells. CD8 memory T cells generated by antigen delivered with both aluminum salts and MPL provided significant protection from influenza A challenge. These adjuvants could be used in human vaccines to prime protective memory CD8 T cells.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigen Presentation; Antigens, Surface; Apoptosis Regulatory Proteins; Cattle; Cytokines; Female; Humans; Immunologic Memory; Influenza A virus; Interleukin-6; Lipid A; Mice; Mice, Knockout; Mice, Transgenic; Nucleocapsid Proteins; Orthomyxoviridae Infections; Ovalbumin; Programmed Cell Death 1 Receptor; RNA-Binding Proteins; Serum Albumin, Bovine; T-Lymphocytes, Cytotoxic; Vaccines, Subunit; Viral Core Proteins

The defective ribosomal product (DRiP) hypothesis of endogenous Ag processing posits that rapidly degraded forms of nascent proteins are a major source of peptide ligands for MHC class I molecules. Although there is broad experimental support for the DRiP hypothesis, careful kinetic analysis of the generation of defined peptide class I complexes has been limited to studies of recombinant vaccinia viruses expressing genes derived from other organisms. In this study, we show that insertion of the SIINFEKL peptide into the stalk of influenza A virus neuraminidase (NA) does not detectably modify NA folding, degradation, transport, or sp. act. when expressed in its natural context of influenza A virus infection. Using the 25-D1.16 mAb specific for K(b)-SIINFEKL to precisely quantitate cell surface complexes by flow cytometry, we demonstrate that SIINFEKL is generated in complete lockstep with initiation and abrogation of NA biosynthesis in both L-K(b) fibroblast cells and DC2.4 dendritic/monocyte cells. SIINFEKL presentation requires active proteasomes and TAP, consistent with its generation from a cytosolic DRiP pool. From the difference in the shutoff kinetics of K(b)-SIINFEKL complex expression following protein synthesis versus proteasome inhibition, we estimate that the t(1/2) of the biosynthetic source of NA peptide is approximately 5 min. These observations extend the relevance of the DRiP hypothesis to viral proteins generated in their natural context.

Topics: Amino Acid Sequence; Animals; Antigen Presentation; Antigens, Viral; Cell Line; Dendritic Cells; Dogs; Enzyme Activation; Enzyme Stability; Epitopes; Fibroblasts; H-2 Antigens; Influenza A Virus, H1N1 Subtype; L Cells; Mice; Molecular Sequence Data; Monocytes; Neuraminidase; Orthomyxoviridae Infections; Ovalbumin; Peptide Fragments; Protein Biosynthesis; Protein Folding; Protein Transport; Ribosomal Proteins

CD8(+) T lymphocytes mediate the immune response to viruses, intracellular bacteria, protozoan parasites, and tumors. We provide evidence that the transcription factor Bcl11b/Ctip2 controls hallmark features of CD8(+) T cell immunity, specifically antigen (Ag)-dependent clonal expansion and cytolytic activity. The reduced clonal expansion in the absence of Bcl11b was caused by altered proliferation during the expansion phase, with survival remaining unaffected. Two genes with critical roles in TCR signaling were deregulated in Bcl11b-deficient CD8(+) T cells, CD8 coreceptor and Plcgamma1, both of which may contribute to the impaired responsiveness. Bcl11b was found to bind the E8I, E8IV, and E8V, but not E8II or E8III, enhancers. Thus, Bcl11b is one of the transcription factors implicated in the maintenance of optimal CD8 coreceptor expression in peripheral CD8(+) T cells through association with specific enhancers. Short-lived Klrg1(hi)CD127(lo) effector CD8(+) T cells were formed during the course of infection in the absence of Bcl11b, albeit in smaller numbers, and their Ag-specific cytolytic activity on a per-cell basis was altered, which was associated with reduced granzyme B and perforin.

Topics: Adoptive Transfer; Animals; Antigen Presentation; Apoptosis; CD4-Positive T-Lymphocytes; CD8 Antigens; CD8-Positive T-Lymphocytes; Cell Proliferation; Clone Cells; Cytotoxicity, Immunologic; Enhancer Elements, Genetic; Gene Expression Regulation; Granzymes; Listeria monocytogenes; Listeriosis; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Orthomyxoviridae Infections; Ovalbumin; Peptide Fragments; Phospholipase C gamma; Pore Forming Cytotoxic Proteins; Protein Binding; Receptors, Antigen, T-Cell; Repressor Proteins; Signal Transduction; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; Tumor Suppressor Proteins; ZAP-70 Protein-Tyrosine Kinase

TCR repertoire diversity can influence the efficacy of CD8(+) T-cell populations, with greater breadth eliciting better protection. We analyzed TCR beta diversity and functional capacity for influenza-specific CD8(+) T cells expressing a single TCR alpha chain. Mice (A7) transgenic for the H2K(b)OVA(257-264)-specific V alpha 2.7 TCR were challenged with influenza to determine how fixing this "irrelevant" TCR alpha affects the "public" and restricted D(b)NP(366) (+)CD8(+) versus the "private" and diverse D(b)PA(224) (+)CD8(+) responses. Though both D(b)NP(366) (+)CD8(+) and D(b)PA(224) (+)CD8(+) sets are generated in virus-primed A7 mice, the constrained D(b)NP(366) (+)CD8(+) population lacked the characteristic, public TCRV beta 8.3, and consequently was reduced in magnitude and pMHC-I avidity. For the more diverse D(b)PA(224) (+)CD8(+) T cells, this particular forcing led to a narrowing and higher TCR beta conservation of the dominant V beta 7, though the responses were of comparable magnitude to C57BL/6J controls. Interestingly, although both the TCR beta diversity and the cytokine profiles were reduced for the D(b)NP(366) (+)CD8(+) and D(b)PA(224) (+)CD8(+) sets in spleen, the latter measure of polyfunctionality was comparable for T cells recovered from the infected lungs of A7 and control mice. Even "sub-optimal" TCR alpha beta pairs can operate effectively when exposed in a milieu of high virus load. Thus, TCR beta diversity is important for optimal TCR alpha beta pairing and function when TCR alpha is limiting.

Topics: Animals; CD8-Positive T-Lymphocytes; Cells, Cultured; Cytokines; Genes, T-Cell Receptor beta; Genetic Variation; H-2 Antigens; Histocompatibility Antigen H-2D; Influenza A virus; Lung; Mice; Mice, Inbred C57BL; Mice, Transgenic; Orthomyxoviridae Infections; Ovalbumin; Peptide Fragments; Protein Multimerization; Receptors, Antigen, T-Cell, alpha-beta; Viral Core Proteins

Priming of naive CD8(+) T cells by pathogens or vaccines generally involves their interaction with Ag-loaded dendritic cells (DCs) in the context of an inflamed lymph node. Lymph node activation fosters DC and T cell encounters and subsequently provides newly primed T cells with nurturing conditions. We dissected these two aspects by infusing in vitro primed CD8(+) T cells into naive recipient mice harboring a single activated lymph node and comparing the fate of these T cells with those infused into control recipients. Brief (20 h) in vitro priming empowered the T cells to expand in vivo without further Ag stimulation. This primary response was not affected by the presence or absence of a nonspecifically activated lymph node. In contrast, in vivo antigenic challenge after contraction of the primary response resulted in significantly stronger secondary T cell responses in mice harboring activated lymph nodes, demonstrating that the availability of an activated lymph node supported the generation of T cell memory in an Ag-unrelated manner. The presence of an activated lymph node during the expansion and contraction phase of the primary response did not endow T cells with an instructional program for increased survival or secondary expansion, but primarily served to conserve increased numbers of T cells.

Topics: Animals; Antigen Presentation; CD8-Positive T-Lymphocytes; Cell Differentiation; Cells, Cultured; Coculture Techniques; Immunologic Memory; Inflammation; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Orthomyxoviridae Infections; Ovalbumin; Peptide Fragments

There is a clear link between obesity and metabolic disorders; however, little is known about the effect of obesity on immune function, particularly during an infection. We have previously reported that diet-induced obese mice are more susceptible to morbidity and mortality during influenza infection than lean mice. Obese mice displayed aberrant innate immune responses characterized by minimal induction of interferon (IFN)-alpha/beta, delayed expression of pro-inflammatory cytokines and chemokines, and impaired natural killer cell cytotoxicity. To further examine the abnormal immune response of diet-induced obese mice, we analysed the cellularity of their lungs during influenza virus infection. We found delayed mononuclear cell entry with a marked decrease in dendritic cells (DCs) throughout the infection. Given the critical role of the DC in activating the cell-mediated immune response, we also analysed the functional capacity of DCs from obese mice. We found that, while obesity did not interfere with antigen uptake and migration, it did impair DC antigen presentation. This was probably attributable to an altered cytokine milieu, as interleukin (IL)-2, IL-12, and IL-6 were differentially regulated in the obese mice. Overall, this did not impact the total number of virus-specific CD8(+) T cells that were elicited, but did affect the number and frequency of CD3(+) and CD8(+) T cells in the lung. Thus, obesity interferes with cellular responses during influenza infection, leading to alterations in the T-cell population that ultimately may be detrimental to the host.

Topics: Animals; Antigen Presentation; CD8-Positive T-Lymphocytes; Cells, Cultured; Dendritic Cells; Diet; Immunity, Innate; Influenza A virus; Interleukin-6; Lung; Lymph Nodes; Mice; Mice, Inbred C57BL; Mice, Obese; Obesity; Orthomyxoviridae Infections; Ovalbumin

Antigen expressed as MHC Class I glycoprotein (pMHCI) complexes on dendritic cells is the primary driver of CD8(+) T cell clonal expansion and differentiation. As we seek to define the molecular differences between acutely stimulated cytotoxic T lymphocyte (CTL) effectors and long-lived memory T cells, it is essential that we understand the duration of in vivo pMHCI persistence. Although infectious influenza A virus is readily cleared by mammalian hosts, that does not necessarily mean that all influenza antigen is totally eliminated. An exhaustive series of carefully controlled adoptive transfer experiments using 3 different carboxy fluorescein diacetate succinimidyl ester-labeled T cell receptor-transgenic CTL populations and a spectrum of genetically engineered and wild-type influenza A viruses provided no evidence for pMHCI persistence over the 30-60-d interval after virus challenge. Molecular profiles identified in antigen-specific T cells at this time may thus be considered to reflect established immunologic memory and not recent CTL activation from a persistent pMHCI pool.

Topics: Animals; Antigen Presentation; CD11c Antigen; CD8-Positive T-Lymphocytes; Cell Movement; Dendritic Cells; Epitopes; Female; Fluoresceins; Histocompatibility Antigens Class I; Inflammation; Influenza A virus; Kinetics; Lung; Lymphocyte Activation; Lymphocyte Count; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Ovalbumin; Phenotype; Succinimides; T-Lymphocytes, Cytotoxic; Time Factors

The magnitude of antigen-specific CD8+ T cell responses is not fixed but correlates with the severity of infection. Although by definition T cell response size is the product of both the capacity to recruit naïve T cells (clonal selection) and their subsequent proliferation (clonal expansion), it remains undefined how these two factors regulate antigen-specific T cell responses. We determined the relative contribution of recruitment and expansion by labeling naïve T cells with unique genetic tags and transferring them into mice. Under disparate infection conditions with different pathogens and doses, recruitment of antigen-specific T cells was near constant and close to complete. Thus, naïve T cell recruitment is highly efficient, and the magnitude of antigen-specific CD8+ T cell responses is primarily controlled by clonal expansion.

Topics: Adoptive Transfer; Ampicillin; Animals; Anti-Bacterial Agents; Antigens; Antigens, Bacterial; Antigens, Viral; CD8-Positive T-Lymphocytes; Dendritic Cells; Epitopes; Genes, T-Cell Receptor alpha; Genes, T-Cell Receptor beta; Influenza A virus; Listeriosis; Lymphocyte Activation; Lymphocyte Count; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Sequence Data; Orthomyxoviridae Infections; Ovalbumin; Receptors, Antigen, T-Cell, alpha-beta; Spleen; Vaccinia; Virus Diseases

The type I interferon (IFN) system is critical for protecting the mammalian host from numerous virus infections and plays a key role in shaping the antiviral adaptive immune response. In this report, the importance of type I IFN signaling was assessed in a mouse model of alphavirus-induced humoral immune induction. Venezuelan equine encephalitis virus replicon particles (VRP) expressing the hemagglutinin (HA) gene from influenza virus (HA-VRP) were used to vaccinate both wildtype (wt) and IFN alpha/beta receptor knockout (RKO) mice. HA-VRP vaccination induced equivalent levels of flu-specific systemic IgG, mucosal IgG, and systemic IgA antibodies in both wt and IFN RKO mice. In contrast, HA-VRP vaccination of IFN RKO mice failed to induce significant levels of flu-specific mucosal IgA antibodies at multiple mucosal surfaces. In the VRP adjuvant system, co-delivery of null VRP with ovalbumin (OVA) protein significantly increased the levels of OVA-specific serum IgG, fecal IgG, and fecal IgA antibodies in both wt and RKO mice, suggesting that type I IFN signaling plays a less significant role in the VRP adjuvant effect. Taken together, these results suggest that (1) at least in regard to IFN signaling, the mechanisms which regulate alphavirus-induced immunity differ when VRP are utilized as expression vectors as opposed to adjuvants, and (2) type I IFN signaling is required for the induction of mucosal IgA antibodies directed against VRP-expressed antigen. These results shed new light on the regulatory networks which promote immune induction, and specifically mucosal immune induction, with alphavirus vaccine vectors.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Viral; Antigens, Viral; Encephalitis Virus, Venezuelan Equine; Enzyme-Linked Immunosorbent Assay; Genes, Viral; Hemagglutinin Glycoproteins, Influenza Virus; Immunity, Mucosal; Immunoglobulin A; Immunoglobulin G; Influenza A virus; Interferon Type I; Mice; Mice, Knockout; Orthomyxoviridae Infections; Ovalbumin; Replicon; Viral Vaccines

Effector memory T (Tem) cells are essential mediators of autoimmune disease and delayed-type hypersensitivity (DTH), a convenient model for two-photon imaging of Tem cell participation in an inflammatory response. Shortly (3 hr) after entry into antigen-primed ear tissue, Tem cells stably attached to antigen-bearing antigen-presenting cells (APCs). After 24 hr, enlarged Tem cells were highly motile along collagen fibers and continued to migrate rapidly for 18 hr. Tem cells rely on voltage-gated Kv1.3 potassium channels to regulate calcium signaling. ShK-186, a specific Kv1.3 blocker, inhibited DTH and suppressed Tem cell enlargement and motility in inflamed tissue but had no effect on homing to or motility in lymph nodes of naive and central memory T (Tcm) cells. ShK-186 effectively treated disease in a rat model of multiple sclerosis. These results demonstrate a requirement for Kv1.3 channels in Tem cells during an inflammatory immune response in peripheral tissues. Targeting Kv1.3 allows for effector memory responses to be suppressed while central memory responses remain intact.

Topics: Animals; Antigen-Presenting Cells; Cell Movement; Chlamydia Infections; Chlamydia trachomatis; Collagen; Encephalomyelitis, Autoimmune, Experimental; Female; Hypersensitivity, Delayed; Immunologic Memory; Kv1.3 Potassium Channel; Lymph Nodes; Lymphocyte Activation; Orthomyxoviridae; Orthomyxoviridae Infections; Ovalbumin; Potassium Channel Blockers; Proteins; Rats; Rats, Inbred Lew; Receptors, CCR7; T-Lymphocytes

In our previous study, OVA conjugated on the surface of a liposome, we termed Oleoyl liposome, which consisted of dioleoyl phosphatidyl choline, dioleoyl phosphatidyl ethanolamine, dioleoyl phosphatidyl glycerol acid and cholesterol in a 4:3:7:2 molar ratio, induced OVA-specific IgG antibody production but not OVA-specific IgE antibody production that is detrimental to the host. Furthermore, OVA(257-264)-Oleoyl liposome elicited CTL responses in the presence of CpG and rejected E.G7 tumors in mice. In this study we tested whether a peptide-Oleoyl liposome conjugates are capable of inducing protection against viral growth. Subcutaneous inoculation of NP(366-374)-Oleoyl liposome with CpG inhibited growth of influenza viruses in lungs of mice. Thus, surface-linked liposomal peptide might serve as an effective vaccine without detrimental effects in the presence of immune potentiators.

Topics: Adjuvants, Immunologic; Animals; Cell Line; Dogs; Enzyme-Linked Immunosorbent Assay; Influenza A Virus, H3N2 Subtype; Influenza Vaccines; Ligands; Liposomes; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Ovalbumin; Spleen; T-Lymphocytes, Cytotoxic; Toll-Like Receptors; Vaccines, Subunit; Viral Plaque Assay

A reverse genetics strategy was used to insert the OVA peptide (amino acid sequence SIINFEKL; OVA(257-264)) into the neuraminidase stalk of both the A/PR8 (H1N1) and A/HKx31 (H3N2) influenza A viruses. Initial characterization determined that K(b)OVA257 is presented on targets infected with PR8-OVA and HK-OVA without significantly altering D(b) nucleoprotein (NP)366 presentation. There were similar levels of K(b)OVA257- and D(b)NP366-specific CTL expansion following both primary and secondary intranasal challenge. Interestingly, while variable, the presence of the immunodominant K(b)OVA257-specific response resulted in diminished D(b) acidic polymerase224- and K(b) basic polymerase subunit 1(703)-, but not D(b)NP366-specific responses and didn't alter endogenous influenza A virus-specific immunodominance hierarchies. However, challenging PR8-OVA-primed mice with HK-OVA via the i.p. route, and thereby limiting Ag dose, led to a reduction in the magnitude of all the influenza A virus-specific responses measured. A similar reduction in CTL response to native epitopes was also seen following primary respiratory HK-OVA infection of mice that received substantial numbers of K(b)OVA257-specific TCR transgenic T cells. Thus, during the course of infection, the generation of individual virus-specific CTL responses is independently regulated. However, in cases in which Ag is limiting, or high precursor frequency, the presence of immunodominant CTL responses can impact on the magnitude of other specific populations. Therefore, depending on both the size of the T cell precursor pool and the mode of Ag presentation, the addition of a major epitope can diminish the size of endogenous, influenza-specific CD8+ T cell responses, although never to the point that these are totally compromised.

Topics: Animals; Antigens, Viral; CD8-Positive T-Lymphocytes; Epitopes, T-Lymphocyte; Female; Influenza A virus; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Lung; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Ovalbumin; Peptide Fragments

Costimulatory signals from dendritic cells (DCs) are required for naive T cells to respond to antigenic stimulation. To what extent DCs reactivate memory T cells during recall responses is not known. Here, an in vivo depletion system has been used to analyze the role of DCs in reactivating CD8 memory T cells during recall responses to three different microbial infections. We show a profound decrease in the numbers of responding memory CD8 T cells in both lymphoid and nonlymphoid tissues during the recall responses to infection with vesicular stomatitis virus, Listeria monocytogenes (Lm), or influenza virus. These data show that interaction with DCs is a major mechanism driving T cell reactivation in vivo, even during a tissue-specific infection of the respiratory tract.

Topics: Animals; CD8-Positive T-Lymphocytes; Chimera; Dendritic Cells; Green Fluorescent Proteins; Heparin-binding EGF-like Growth Factor; Immunologic Memory; Infections; Intercellular Signaling Peptides and Proteins; Listeriosis; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Orthomyxoviridae Infections; Ovalbumin; Receptors, Cell Surface; Rhabdoviridae Infections; Signal Transduction; Vesicular stomatitis Indiana virus

To track epitope-specific CD4(+) T cells at a single-cell level during influenza infection, the MHC class II-restricted OVA(323-339) epitope was engineered into the neuraminidase stalk of influenza/A/WSN, creating a surrogate viral antigen. The recombinant virus, influenza A/WSN/OVA(II), replicated well, was cleared normally, and stimulated both wild-type and DO11.10 or OT-II TCR transgenic OVA-specific CD4(+) T cells. OVA-specific CD4 T cells proliferated during infection only when the OVA epitope was present. However, previously primed (but not naive) transgenic CD4(+) T cells were recruited to the infected lung both in the presence and absence of the OVA(323-339) epitope. These data show that, when primed, CD4(+) T cells may traffic to the lung in the absence of antigen, but do not proliferate. These results also document a useful tool for the study of CD4 T cells in influenza infection.

Topics: Adoptive Transfer; Amino Acid Sequence; Animals; CD4-Positive T-Lymphocytes; Cell Line; Disease Models, Animal; Dogs; Influenza A virus; Kidney; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Sequence Data; Orthomyxoviridae Infections; Ovalbumin; Peptide Fragments; Viral Plaque Assay

alpha-Galactosylceramide (alpha-GalCer) is a ligand of invariant Valpha14+ NKT cells and is presented by CD1d molecule on APC. NKT cells produce a large amount of Th1 and Th2 cytokines in response to alpha-GalCer-presented APC. In this study, we assessed whether alpha-GalCer could act as an effective nasal vaccine adjuvant for mucosal vaccine that would be capable of inducing systemic as well as mucosal immune responses. When alpha-GalCer was administered with OVA via the intranasal route to C57BL/6 and BALB/c mice, significant OVA-specific mucosal secretory IgA, systemic IgG, and CTL responses were induced with mixed Th1 and Th2 cytokine profiles seen in both strains of mice. Interestingly, as BALB/c mice were intranasally immunized with PR8 hemagglutinin Ag isolated from influenza virus A/PR/8/34 together with alpha-GalCer, significant protection was afforded against influenza viral infection. When alpha-GalCer was coimmunized with a replication-deficient live adenovirus to BALB/c mice, it significantly induced both humoral and cellular immune responses. In addition, intranasal administration of OVA with alpha-GalCer showed complete protection against EG7 tumor challenge in C57BL/6. The adjuvant effects induced by intranasal coadministration with alpha-GalCer were blocked in CD1d-/- mice, indicating that the immune responses were exclusively mediated by CD1d molecule on APC. Most interestingly, intranasally coadministered alpha-GalCer activated naive T cells and triggered them to differentiate into functional effector T cells when CFSE-labeled OT-1 cells were adoptively transferred into syngeneic mice. Overall, our results are the first to show that alpha-GalCer can act as a nasal vaccine adjuvant inducing protective immune responses against viral infections and tumors.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antigens, CD1; Antigens, CD1d; Cancer Vaccines; Cytokines; Defective Viruses; Female; Galactosylceramides; Immunoglobulin A, Secretory; Immunoglobulin G; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasms, Experimental; Orthomyxoviridae Infections; Ovalbumin; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Viral Vaccines

Respiratory viral infections in early childhood may interact with the immune system and modify allergen sensitization and/or allergic manifestations. In mice, respiratory syncytial virus (RSV) infection during allergic provocation aggravates the allergic T helper (Th) 2 immune response, characterized by the production of IL-4, IL-5, and IL-13, and inflammatory infiltrates. However, it is unclear whether the RSV-enhanced respiratory allergic response is a result of non-specific virus-induced damage of the lung, or virus-specific immune responses.. In the present study we investigated whether RSV, pneumonia virus of mice (PVM) and influenza A virus similarly affect the allergic response.. BALB/c mice were sensitized and challenged with ovalbumin (OVA), and inoculated with virus during the challenge period. Pulmonary inflammation, lung cytokine mRNA responses, and IgE production in serum were assessed after the last OVA-challenge.. Like RSV, PVM enhanced the OVA-induced pulmonary IL-4, IL-5, and IL-13 mRNA expression, which was associated with enhanced perivascular inflammation. In addition, PVM increased the influx of eosinophils in lung tissue. In contrast, influenza virus decreased the Th2 cytokine mRNA expression in the lungs. However, like PVM, influenza virus enhanced the pulmonary eosinophilic infiltration in OVA-allergic mice.. The Paramyxoviruses RSV and PVM both are able to enhance the allergic Th2 cytokine response and perivascular inflammation in BALB/c mice, while the Orthomyxovirus influenza A is not.

Topics: Animals; Female; Hypersensitivity; Immunoglobulin E; Influenza A virus; Interleukin-13; Interleukin-4; Interleukin-5; Lung; Mice; Mice, Inbred BALB C; Murine pneumonia virus; Orthomyxoviridae Infections; Ovalbumin; Pneumovirus Infections; Pulmonary Eosinophilia; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; RNA, Messenger; Virus Diseases

Respiratory viral infections are a leading cause of the hospitalization of asthmatics, however, the cellular immunological interactions which underlie these two diseases remain elusive.. We sought to characterize the effect influenza viral infection has on allergic airway inflammation and to identify the cellular pathways involved.. We have used an ovalbumin (OVA) model of allergic airway inflammation, which involves sensitization of animals with OVA adsorbed in alum adjuvant followed by an intranasal challenge with OVA in phosphate-buffered saline. To study T cell recruitment into the lung, we adoptively transferred in vitro activated T cell receptor-transgenic T cells, which were subsequently identified by fluorescence-activated cell sorting (FACS) analysis. In addition, to study in vivo dendritic cell (DC) migration, we administered fluorescently labelled dextran and identified DCs that had phagocytosed it by FACS analysis.. We found that different stages of influenza infection had contrasting effects upon the outcome of OVA-induced allergic airway inflammation. The allergic response against OVA was exacerbated during the acute stage of influenza infection; however, mice were protected against the development of airway eosinophilia at late time-points following infection. We investigated the mechanisms responsible for the virus-induced exacerbation and found that the response was partially independent of IL-4 and that there was increased delivery of inhaled allergens to the draining lymph node during the acute stage of the infection. In addition, virus-induced inflammation in the lung and draining lymph node resulted in the non-specific recruitment of circulating allergen-specific effector/memory cells.. In addition to virus-mediated damage to the lung and airways, influenza viral infection can also enhance unrelated local allergic responses.

Topics: Acute Disease; Allergens; Animals; Asthma; Bronchi; Flow Cytometry; Immunologic Memory; Mice; Mice, Inbred C57BL; Mice, Transgenic; Models, Animal; Orthomyxoviridae Infections; Ovalbumin; Plethysmography; Receptors, Antigen, T-Cell; Th2 Cells

Most infections with respiratory viruses induce Th1 responses characterized by the generation of Th1 and CD8(+) T cells secreting IFN-gamma, which in turn have been shown to inhibit the development of Th2 cells. Therefore, it could be expected that respiratory viral infections mediate protection against asthma. However, the opposite seems to be true, because viral infections are often associated with the exacerbation of asthma. For this reason, we investigated what effect an influenza A (flu) virus infection has on the development of asthma. We found that flu infection 1, 3, 6, or 9 wk before allergen airway challenge resulted in a strong suppression of allergen-induced airway eosinophilia. This effect was associated with strongly reduced numbers of Th2 cells in the airways and was not observed in IFN-gamma- or IL-12 p35-deficient mice. Mice infected with flu virus and immunized with OVA showed decreased IL-5 and increased IFN-gamma, eotaxin/CC chemokine ligand (CCL)11, RANTES/CCL5, and monocyte chemoattractant protein-1/CCL2 levels in the bronchoalveolar lavage fluid, and increased airway hyperreactivity compared with OVA-immunized mice. These results suggest that the flu virus infection reduced airway eosinophilia by inducing Th1 responses, which lead to the inefficient recruitment of Th2 cells into the airways. However, OVA-specific IgE and IgG1 serum levels, blood eosinophilia, and goblet cell metaplasia in the lung were not reduced by the flu infection. Flu virus infection also directly induced AHR and goblet cell metaplasia. Taken together, our results show that flu virus infections can induce, exacerbate, and suppress features of asthmatic disease in mice.

Topics: Allergens; Animals; Bronchial Hyperreactivity; Cell Migration Inhibition; Cell Movement; Cells, Cultured; Chemokine CCL11; Chemokine CCL2; Chemokine CCL5; Chemokines, CC; Down-Regulation; Epitopes, T-Lymphocyte; Goblet Cells; Influenza A virus; Interferon-gamma; Interleukin-5; Lung; Lymphocyte Count; Lymphopenia; Metaplasia; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nippostrongylus; Orthomyxoviridae Infections; Ovalbumin; Peptide Fragments; Pulmonary Eosinophilia; Strongylida Infections; Th2 Cells; Up-Regulation

We evaluated the proposal that during microbial infection, dendritic cells (DCs) undergo maturation and present a mixture of peptides derived from the microbe as well as harmless environmental antigens. Mice were exposed to an aerosol of endotoxin free ovalbumin (OVA) in the absence or presence of influenza virus. In its absence, OVA failed to induce B and T cell responses and even tolerized, but with influenza, OVA-specific antibodies and CD8+ cytolytic T lymphocytes developed. With or without infection, OVA was presented selectively in the draining mediastinal lymph nodes, as assessed by the comparable proliferation of infused, CD8+ and CD4+, TCR transgenic T cells. In the absence of influenza, these OVA-specific T cells produced little IL-2, IL-4, IL-10, and IFN-gamma, but with infection, both CD4+ and CD8+ T cells made high levels of IL-2 and IFN-gamma. The OVA plus influenza-treated mice also showed accelerated recovery to a challenge with recombinant vaccinia OVA virus. CD11c+ DCs from the mediastinal lymph nodes of infected mice selectively stimulated both OVA- and influenza-specific T cells and underwent maturation, with higher levels of MHC class II, CD80, and CD86 molecules. The relatively slow (2-3 d) kinetics of maturation correlated closely to the time at which OVA inhalation elicited specific antibodies. Therefore respiratory infection can induce DC maturation and simultaneously B and T cell immunity to an innocuous antigen inhaled concurrently.

Topics: Animals; Antibody Formation; Antigen Presentation; Antigens, Viral; CD11c Antigen; Dendritic Cells; Female; Immune Tolerance; Lymph Nodes; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Ovalbumin; T-Lymphocytes

To study which phase of viral infection promotes antigen sensitization via the airway and which type of antigen-presenting cells contributes to antigen sensitization, BALB/c mice were sensitized by inhalation of ovalbumin (OA) during the acute phase or the recovery phase of influenza A virus infection, and then 3 weeks later animals were challenged with OA. The numbers of eosinophils and lymphocytes, the amounts of interleukin-4 (IL-4) and IL-5 in the bronchoalveolar lavage fluid, and the serum levels of OA-specific immunoglobulin G1 (IgG1) and IgE increased in mice sensitized during the acute phase (acute phase group), while a high level of gamma interferon production was detected in those sensitized during the recovery phase (recovery phase group). In the acute phase group, both major histocompatibility complex class II molecules and CD11c were strongly stained on the bronchial epithelium; in the recovery phase group, however, neither molecule was detected. OA-capturing dendritic cells (DCs) migrated to the regional lymph nodes, and a small number of OA-capturing macrophages were also observed in the lymph nodes of the acute phase group. In the recovery group, however, no OA-capturing DCs were detected in either the lungs or the lymph nodes, while OA-capturing macrophages were observed in the lymph nodes. These results indicate that the timing of antigen sensitization after viral infection determines the type of immune response.

Topics: Animals; Bronchoalveolar Lavage Fluid; CD11 Antigens; Cytokines; Dendritic Cells; Immunoglobulin E; Immunoglobulin G; Immunohistochemistry; Influenza A virus; Lung; Male; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Ovalbumin; Time Factors

The role of Ag in the recruitment and localization of naive, acutely activated, and memory CD8(+) T cells to the lung during influenza infection was explored using TCR-transgenic (Tg) mice. Naive, Thy1.2(+)CD8(+) OT-I TCR-Tg cells were primed and recruited to the lung after transfer into congenic Thy1.1(+) recipients challenged with a genetically engineered influenza virus (influenza A/WSN/33 (WSN)-OVA(I)) containing the K(b) restricted OVA(257-264) epitope (siinfekl) in the viral neuraminidase stalk. However, if the transferred animals were infected with a similar influenza virus that expressed an irrelevant K(b) epitope (WSN-PEPII), no TCR-Tg T cells were detectable in the lung, although they were easily visible in the lymphoid organs. Conversely, there were substantial numbers of OT-I cells found in the lungs of WSN-PEPII-infected mice when the animals had been previously, or were concurrently, infected with a recombinant vaccinia virus expressing OVA. Similar results were obtained with nontransgenic populations of memory CD8(+) T cells reactive to a murine gamma-herpesvirus-68 Ag. Interestingly, the primary host response to the immunodominant influenza nucleoprotein epitope was not affected by the presence of memory or recently activated OT-I T cells. Thus, although Ag is required to activate the T cells, the subsequent localization of T cells to the lung during a virus infection is a property of recently activated and memory T cells and is not necessarily driven by Ag in the lung.

Topics: Adoptive Transfer; Animals; Antigens, Viral; CD8-Positive T-Lymphocytes; Cell Movement; Cells, Cultured; DNA, Recombinant; DNA, Viral; Egg Proteins; Female; Genes, T-Cell Receptor; Immunologic Memory; Influenza A virus; Lung; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Orthomyxoviridae Infections; Ovalbumin; Peptide Fragments; Pneumonia, Viral; Rhadinovirus

To study the mechanisms responsible for enhanced sensitization of inhaled antigen in respiratory viral infections, we examined the contribution of dendritic cells (DC) and T lymphocytes to the development of inhalation sensitization during infection with influenza A virus in mice. BALB/c mice were sensitized by inhalation of ovalbumin (OA) from 3 to 7 days after the inoculation with influenza A virus, and were challenged with OA 3 weeks later. Airway responsiveness and serum OA-specific IgE were increased. The numbers of eosinophils and CD4(+) and CD8(+) T cells in bronchoalveolar lavage fluid were also increased. These changes were not observed in animals only sensitized with OA or only inoculated with the virus. In animals only inoculated with the virus, DC were immunohistochemically detected on the bronchial epithelium on days 2-5. With OA inhalation after virus inoculation, DC with high expression of MHC class II were retained for 5 weeks. These results show that influenza virus infection induces the migration of DC to the bronchial epithelium, and that simultaneous inhalation of antigen causes the loading of antigen-peptide / class II molecule complex on DC. Thus, the migration of DC in viral infection may play some role in the augmentation of antigen sensitization.

Topics: Animals; Antigen Presentation; Bronchoalveolar Lavage Fluid; Cytokines; Dendritic Cells; Immunization; Immunoglobulin E; Immunophenotyping; Influenza A virus; Interferon-gamma; Lung; Male; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Ovalbumin

Virus infections frequently exacerbate asthma, and in some cases may even precipitate its onset. Although this association is well known, experimental investigation has been hampered by the lack of adequate models.. The effects of acute respiratory virus infection on sensitization to aereoallergen were investigated in this study.. Nebulized ovalbumin was used as an aeroantigen in normal mice, and in those infected with respiratory syncytial virus or influenza A.. Both viruses caused transient illness. Ovalbumin inhalation did not induce specific serum antibodies unless the mice were infected at the time of nebulization. In exposed uninfected mice cutaneous challenge with ovalbumin caused no response, but caused acute systemic illness and collapse if previous pulmonary exposure had occurred during respiratory infection. Mice that collapsed in response to cutaneous ovalbumin were found to have IgG1 specific to ovalbumin that was not found in the other mice. Intracellular cytokine staining of splenocyte cultures showed ovalbumin-specific production of IL-4 was enhanced by virus infection during exposure. In CD8+ T cells, ovalbumin-specific interferon-gamma production was also enhanced by co-infection with influenza. Both viruses were equally associated with the induction of anaphylaxis.. These results show that infection with respiratory viruses powerfully augments cellular and humoral immune responses to aeroantigen and provide an experimental model that allows such effects to be investigated.

Topics: Administration, Inhalation; Allergens; Anaphylaxis; Animals; Bronchial Hyperreactivity; Female; Flow Cytometry; Immunoglobulin E; Immunoglobulin G; Influenza A virus; Injections, Intradermal; Mice; Orthomyxoviridae Infections; Ovalbumin; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Respiratory Tract Infections; Spleen; Weight Loss

Upper respiratory tract viral infections have been reported in clinical studies to serve as risk factors for allergic sensitization. In order to study the relationship linking influenza virus illnesses to development of allergy, murine models of allergen sensitization were previously employed. These models showed that lethal influenza viruses were able to trigger allergen-specific immunoglobulin E (IgE) production and to inhibit tolerance to repeated exposure to aerosolized allergen in the mouse. The disadvantage of these murine models consists in the utilization of virulant and lethal strains of influenza virus. A nonlethal rat-adapted influenza virus (RAIV) host resistance model has been developed in our laboratory. It was used to evaluate the effect of influenza virus infection on IgE responses to inhaled ovalbumin (OA) in the rat. The high IgE-responder Brown-Norway (BN) rat was chosen for further study after comparing the IgE response to OA in Fischer 344 (F344) and BN rats. On d 1, BN rats were sensitized by administration of 1 mg OA subcutaneously alone or together with aluminum hydroxide (200 mg) and Bordetella pertussis (15 x 10(9) killed bacilli per rat in 1 ml), or only received saline. Rats were either infected with RAIV or sham-infected on d 0 (24 h prior to sensitization) or on d 15, 17, or 57. Rats were exposed for 3 min to aerosolized OA (OA 3% in phosphate-buffered saline) every week, starting on d 18. Serum OA-specific IgE was evaluated by reverse enzyme-linked immunosorbent assay (ELISA) 3 d after each OA challenge. BN rats elicited a detectable OA-specific IgE response that decreased after repeated aerosol exposures. Influenza virus infection transiently increased the OA-specific IgE response when rats were immunized with OA alone and were infected 1 d prior to the first challenge and also when rats received only saline on d 1, were exposed each week to aerosolized OA, and were infected prior to the seventh challenge. These results, with data previously reported in mice, emphasize the importance of upper respiratory tract viral infection in increasing IgE responses to allergens and may be of importance in human disease.

Topics: Administration, Inhalation; Aerosols; Animals; Disease Models, Animal; Female; Hypersensitivity; Immunoglobulin E; Injections, Subcutaneous; Orthomyxoviridae; Orthomyxoviridae Infections; Ovalbumin; Rats; Rats, Inbred BN; Rats, Inbred F344; Risk Factors; Viral Plaque Assay; Virus Replication

Topics: Animals; Antibody Specificity; Enzyme-Linked Immunosorbent Assay; Germ-Free Life; Immunoglobulin A; Immunoglobulin G; Influenza A virus; Lymphocyte Subsets; Male; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Nasal Mucosa; Orthomyxoviridae Infections; Ovalbumin; Specific Pathogen-Free Organisms

The present study was undertaken to test the effect of sensitization of mice with ovalbumin on the course of influenza virus infection. It was shown that the endogenous interferon level as well as degree of degranulation of lung mast cells were higher in sensitized and influenza virus-infected mice than in infected and nonsensitized ones. Also, lung cells of mice subjected to influenza virus effect in vitro revealed higher degranulation degree.

Topics: Animals; Chickens; Female; Immunization; Interferons; Lung; Male; Mast Cells; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Ovalbumin

An IgE antibody specific to ovalbumin was produced when C3H mice were first infected with influenza A virus before challenge with the aerosolized antigen. No antibody could be detected in those animals without preceding viral infection. Antigen inhalation immediately after virus infection could not induce the IgE antibody. A time lag of at least 2 days was required to sensitize the infected mice with inhaled antigen. The study revealed that allergic sensitization could be elicited only during the acute stage of the infection, but not in the convalescent stage. We concluded therefore that the inflammation of respiratory mucous membrane might allow inhaled antigens to penetrate the barrier, resulting in reaginic antibody production that has the capacity to serve as an allergic response.

Topics: Administration, Intranasal; Aerosols; Animals; Immunoglobulin E; Influenza A virus; Injections, Intraperitoneal; Lung; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Orthomyxoviridae Infections; Ovalbumin; Rats; Respiratory Hypersensitivity