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Carbohydrates often accomplish as cell-surface receptors for microorganisms and influenza virus preferentially binds to sialic acid through the viral haemagglutinin. The virus may attach not only to the epithelium in the airways, but also to the surface ocular epithelium.

Five experiments were performed with the aim of studying two adjuvants named CAF01 and CAF05. They were tested together with a commercial used influenza A vaccine. The first experiment showed that CAF01 had a markedly effect on the influenza A vaccine by increasing the serum vaccine specific IgG antibody production one year after immunization. No effect on the viral clearance of CAF01 was seen before five days after challenge. However, at day five CAF01 showed a significant effect on influenza vaccine by increase of the viral RNA clearance. This indicates that CAF01 has the ability to induce long lasting protection. In the second experiment cross protection abilities of CAF01 were tested. CAF01 showed no indication to stimulate IgG antibody production that was able to bind to a heterologous Hemagglutinin 1 protein. In contrast CAF01 was observed to stimulate IgG antibody production that was able to bind to a heterologous hemagglutinin 3 protein. Furthermore, CAF01 was able to induce the production of serum specific IgG antibody production and increase the viral RNA clearance. The results indicate that CAF01 have ability to induce cross protection between influenza A species. In the third experiment CAF01 showed to induce the vaccine by increase IgG antibody production and the viral RNA clearance. Furthermore CAF01 showed significant difference to the oil emulsion adjuvant indicating that DDA and TDB have an influence on the efficiency of CAF01. The fourth experiment was a pilot experiment for developing, optimizing and obtaining guidelines for experiment five. CAF05 showed promising indication for inducing the efficiency vaccine. In the fifth experiment CAF05 was tested. CAF05 showed to induce the vaccine by increasing the production of serum IgG and IgA antibodies. There was not observed any effect on the viral RNA clearance. CAF05 did not obtain the same efficiency as CAF01. In add

Inflammatory diseases such as type 2 diabetes (T2D) in humans and mice are under the influence of the composition of the gut microbiota (GM). It was previously demonstrated that treating Lepob mice with antibiotics improved glucose tolerance. However, wild type C57BL/6J mice may also exhibit plasma glucose intolerance reminiscent of human T2D. We hypothesized that antibiotic treatment in C57BL/6 mice would have an impact on glucose tolerance without affecting weight and gut immunology. When compared to mice treated with erythromycin or the controls, treatment for five weeks with ampicillin improved glucose tolerance without significantly affecting the weight or the number of gut mucosal regulatory T cells, tolerogenic dendritic cells or T helper cells type 1. 16S rRNA gene based denaturing gradient gel electrophoresis profiles clearly clustered according to treatment and showed that antibiotic treatment reduced GM diversity. It is concluded that antibiotic treatment changes glucose metabolism as well as the composition of the GM in C57BL/6 mice, and that this does not seem to be correlated to weight development in the mice.

Trivalent inactivated vaccines (TIV) against influenza are given to 350 million people every year. Most of these are non-adjuvanted vaccines whose immunogenicity and protective efficacy are considered suboptimal. Commercially available non-adjuvanted TIV are known to elicit mainly a humoral immune response, whereas the induction of cell-mediated immune responses is negligible. Recently, a cationic liposomal adjuvant (dimethyldioctadecylammonium/trehalose 6,6'-dibehenate, CAF01) was developed. CAF01 has proven to enhance both humoral and cell-mediated immune responses to a number of different experimental vaccine candidates. In this study, we compared the immune responses in ferrets to a commercially available TIV with the responses to the same vaccine mixed with the CAF01 adjuvant. Two recently circulating H1N1 viruses were used as challenge to test the vaccine efficacy. CAF01 improved the immunogenicity of the vaccine, with increased influenza-specific IgA and IgG levels. Additionally, CAF01 promoted cellular-mediated immunity as indicated by interferon-gamma expressing lymphocytes, measured by flow cytometry. CAF01 also enhanced the protection conferred by the vaccine by reducing the viral load measured in nasal washes by RT-PCR. Finally, CAF01 allowed for dose-reduction and led to higher levels of protection compared to TIV adjuvanted with a squalene emulsion. The data obtained in this human-relevant challenge model supports the potential of CAF01 in future influenza vaccines.

More than 33 million people were living with HIV worldwide, of these >22 million live in the Sub-Saharan Africa. A vaccine against HIV-1 is the ultimate way of bringing this disastrous disease and its consequences under control. Understanding the neutralization of local strains is important for development of a custom made antibody inducing vaccine. We report an optimized method for full-length env characterization. This tool can be used to develop a panel of env subtypes circulating West Africa. Using such a panel we can gain information about neutralization of a subtype, sub-subtypes and circulating recombinant forms. We have obtained samples from 85 HIV-1 positive patients in a low-resource setting in The Republic of Guinea-Bissau (RGB). Despite of non-optimal storage conditions of samples in RGB viral loads were measured in twenty patients with a success rate of a 100%. We have developed an optimized method for reverse transcription and amplification of full-length env from plasma. Using this RT-semi-nested PCR method, plasma samples with a viral load down to 5,700 copies /ml can be used for full-length amplification of env. In total we have cloned 47 full-length env amplicons from three patients and a cultivated HIV-1 isolate from RGB. Twenty of the 47 full-length amplified amplicons were full-length sequenced. The translated env nucleotide sequences were analyzed for open reading frame, potential N-glycosylations sites and predicted co-receptor tropism. Two CRF02_AG and one CRF02_AG/A3 hybrid were identified from phylogeny and recombination analysis. Two full-length env with an open reading frame was successfully functionally tested in an optimized luciferase reporter gene assay and infectivity was calculated. Another two pseudotype viruses were prepared and are ready for functionality test. The results from this study are of scientific significance as we have developed an optimized method for the design of a panel of full-length env. Pa

Vi inokulerede mink med to forskellige hvalpesygevirus - en dansk (DK91) og en amerikansk (Snyder Hill) for at undersøge virulensen af de to vildtype virus. D91 repræsenterer en cirkulerende europæisk vildtype genotype, mens Snyder Hill repræsenterer de gamle amerikanske virus genotyper isoleret før 1960. Vi foretog kliniske og parakliniske undersøgelser af dyrene efter inokulation og fandt at de to vildtype hvalpesygevirus havde forskellig sygdomsudvikling og grad af påvirkning af immunsystemet. Den danske vildtype gav anledning til et markant mildere sygdomsforløb end den amerikanske Snyer Hill virus. Herefter undersøgte vi hvorvidt vaccination af mink med DNA vacciner baseret på gener fra vaccinestammer inducerer krydsbeskyttelse mod cirkulerende europæiske hvalpesygevirusstammer. Resultaterne fra vores undersøgelse viste, at DNA vaccinerne havde en beskyttende effekt mod udvikling af hvalpesyge. Resultaterne kan få betydning for fremtidige vaccinestrategier mod hvalpesyge forårsaget af nye vildtypevirus., Mink were inoculated with two different Canine distemper viruses (CDV) - a Danish (DK91) virus strain and an American (Snyder Hill) virus strain. DK91 represents a circulating European wildtype genotype, while the Snyder Hill strain represents the American genotypes isolated before 1960. Clinical and paraclinical investigations of the mink were performed after inoculation. The wildtype CDVs investigated provoked marked virulence differences. The Danish wildtype showed a mildly virulent course in our natural host model, in contrast to the acute, severe disease outcome in the Snyder Hill challenged mink. Furthermore, we investigated if DNA vaccine consisting of the genes from vaccine strains induced cross-protection against circulating European CDV. Our results indicate that DNA vaccine-induced immunity protected the mink against disease development. Further work on DNA vaccines against circulating wildtype CDVs could lead to new and safer strategies to control and to prevent distemper.

Patients with diabetes mellitus (DM) have increased platelet activation compared to non-diabetic controls. Platelet hyperreactivity has been associated with adverse cardiovascular outcomes in Type 2 DM, and with diabetic nephropathy. We investigated the relationship between platelet activation and nephropathy in Type 1 DM. Patients with Type 1 DM and diabetic nephropathy (n = 35), age- and sex-matched Type 1 DM patients with persistent normoalbuminuria (n = 51), and healthy age- and sex-matched controls (n = 30) were studied. Platelet surface P-selectin, platelet surface activated GPIIb/IIIa, monocyte-platelet aggregates (MPAs) and neutrophil-platelet aggregates (NPAs) were measured by whole blood flow cytometry as markers of platelet activation. Platelet reactivity was assessed in response to exogenously added ADP and thrombin receptor activating peptide (TRAP). Platelet surface P-selectin (basal and in response to 0.5 or 20 microM ADP) was higher in nephropathy patients compared with normoalbuminuric patients (P = 0.027), and non-diabetic controls (P = 0.0057). NPAs were higher in nephropathy patients compared to normoalbuminuric patients (P = 0.0088). MPAs were higher in nephropathy patients compared to non-diabetic controls (P = 0.0075). There were no differences between groups in activated GPIIb/IIIa or in response to TRAP at any end-point. More patients with nephropathy received aspirin (71.4%) compared to normoalbuminuric patients (27.4%) (P < 0.0001). Type 1 diabetic nephropathy, as compared with normoalbuminuria, is associated with circulating activated platelets and platelet hyperreactivity to ADP, despite the confounding variable of more nephropathy patients receiving aspirin. This platelet activation is likely to contribute to the known increased risk of cardiovascular events in patients with diabetic nephropathy

Glycans terminating in sialic acids serve as receptors for influenza viruses. In this study ferrets were infected with influenza virus A/New Caledonia/20/99, and the in situ localization of sialic acids linked a2-3 and a2-6 in the airways was investigated in infected and non-infected animals by use of sialic acid detecting lectins and a monoclonal antibody towards the Sialyl-Tn antigen. The goblet cells in the bronchi from non-infected ferrets expressed Sialyla2-6Gal glycans, while the seromucinous glands in the submucosa expressed Sialyla2-3Gal glycans. In the infected animals, the surface epithelial cells in some bronchi showed metaplasia and expressed the Sialyl-Tn antigen: Sialyla2-6GalNAc-O-Thr/Ser. The submucosal tracheal glands in these animals showed increased expression of both Sialyla2-3 and Sialyla2-6 epitopes.

The mechanisms behind the in vivo virulence of immunosuppressive wild-type Morbillivirus infections are still not fully understood. To investigate lymphotropism and host responses we have selected the natural host model of canine distemper virus (CDV) infection in mink. This model displays multisystemic infection similar to measles virus (MV) and rinderpest virus (RPV) infections in their susceptible natural hosts. The wild-type CDVs investigated provoked marked virulence differences inducing mild versus marked to severe acute disease. The mildly virulent wild-type induced transient lymphopenia despite the development of massive infection of peripheral blood mononuclear cells (PBMCs) exceeding that determined for the highly virulent wild-type, indicating an inverse relationship between acute virulence and the extent of viremia between the investigated wild-types. Single-cell cytokine production in PBMCs was investigated throughout the acute infections. We observed Th1- and Th2-type cytokine responses beginning in the prodromal phase, and late inflammatory responses were shared between the wild-type infections.

The immunogenicity and efficacy of preventive vaccines against influenza are considered suboptimal and the development of novel influenza vaccination strategies is urgently needed. Commercially available trivalent split vaccines are known to elicit mainly a humoral immune response, whereas the induction of cell-mediated immune responses is negligible. Recently, a cationic liposomal adjuvant (dimethyldioctadecylammonium/trehalose 6,6’-dibehenate, CAF01) was developed, which was proven to enhance both humoral and cell-mediated immune responses of a number of vaccine candidates. In the current study, we compared the immune response in ferrets vaccinated with a commercial influenza split vaccine with the same vaccine mixed with the CAF01 adjuvant and furthermore used two recently circulating H1N1 viruses for the challenge of the animals. We investigated antibody levels in serum and nasal washes by ELISA, as well as IFN-gamma production by peripheral blood lymphocytes by FACS, and virus excretion by RT-PCR. CAF01 improved the immunogenicity of the vaccine, increasing the specific IgA and IgG levels as well as triggering cellular-mediated immunity. The adjuvant also enhanced the protection conferred by the vaccine, reducing the viral load

Karies hos heste er en sygdom, hvis prævalens er steget de sidste 10 år. Man kender i dag intet til det equine immunsvar mod hestens kariesbakterie Streptococcus devriesei (S. devriesei), og kun lidt til hestens sekretoriske immunsystem. Formålet med dette studie er derfor at undersøge molekylevægten for sekretorisk immunoglobulin A (SIgA) i hestespyt, samt den eventuelle rolle, dette antistof kan spille i karies hos hest. Desuden blev tre forskellige anti-hest-IgA-antistoffer evalueret.Spyt, nasalsekret og serum indsamledes fra 36 kontroldyr samt fra 10 heste med nyopdaget karies og 10 heste med tidligere behandlet karies. Der blev desueden udtaget prøver til bakteriologisk undersøgelse fra 24 af kontroldyrene samt fra 6 af hestene med nyopdaget karies og 7 af dem med tidligere behandlet karies karies. Disse prøver blev analyseret for total bakterievækst samt forekomst af S. devriesei. Immunoblotting blev brugt til at (1) evaluere anti-hest-IgA-antistoffer, (2) undersøge for renhed af oprenset SIgA, samt (3) beregne de forskellige molekylevægte. Indhold af total (µg/ml) og S. devriesei-specifik (titre) SIgA blev målt med ELISA. SIgA blev oprenset fra hestespyt ved udfældning med mættet ammoniumsulfat og oprensning på Protein G column. Oprenset SIgA blev brugt som standard i ELISA for total SIgA samt til beregning af molekylevægte.De tre anti-hest-IgA-antistoffer der blev evalueret genkender forskellige former for hestens IgA. De beregnede molekylevægte stemmer overens med hvad der tidligere er publiceret for hest, og IgA i hesteserum var typisk dimerisk. S. devriesei fandtes i 67% af de analyserede prøver, og både fra kontrolheste og fra heste med karies. SIgA specifik for S. devriesei fandtes i 75% af alle spytprøver, hvilket tyder på at denne bakterie inducerer et immunsvar hos hest. Hverken totalkoncentration af SigA eller titre af S. devriesei specifik SIgA korrelerede med kariesstatus.Disse resultater stemmer ikke overens med tidligere studier, hvor SIgA har h, The prevalence of dental caries has increased in the horse population over the last decade. Nothing is known about the immune response to Streptococcus devriesei (S. devriesei), the “equine caries bacterium”, and little is known about the equine secretory immune system in general. Therefore, the molecular weights for secretory immunoglobulin A (SIgA) in horse saliva and its potential role in dental caries were investigated in this study. Furthermore, three anti-horse IgA antibodies were evaluated.Samples of saliva, nasal secretions and serum were obtained from 36 control animals along with 10 horses with new and 10 horses with treated caries. Thirty-seven samples for bacteriological analysis were also taken from 24 of the control animals, and from 6 of those with new and 7 with treated caries. These were analysed for total bacterial growth and growth of S. devriesei. Immunoblotting techniques were used for evaluation of the anti-horse IgA antibodies, determination of purity of the purified SIgA, and molecular weight determinations. Contents of total (µg/ml) and S. devriesei specific (titres) salivary SIgA was determined by ELISA. SIgA was purified from a pool of horse saliva by precipitation with saturated ammonium sulphate and purification on a Protein G column. Purified SIgA was used as standard in the ELISA for total SIgA and for molecular weight determinations.The evaluated anti-horse IgA antibodies recognised different forms of equine IgA. The determined molecular weights correlated well with what has previously been published for horse immunoglobulins. Interestingly, equine serum appeared to contain more dimeric than monomeric IgA. Growth of S. devriesei was detected in 67% of analysed samples, both in control- and caries-positive horses. S. devriesei specific SIgA was detected in 75% of the animals, indicating that these bacteria induce an immune response in horses. Neither levels of total nor S. devriesei specific SIgA correlated with caries status.The prese

Cytotoxic T lymphocytes (CTLs) are able to recognize peptides derived from tumor associated antigens (TAAs) in context of HLA molecules. TAA-derived peptides can be used in vaccines, and clinical trials with cancer patients have in some cases been promising. Therefore, substantial efforts have been invested in identifying TAAs and characterizing the CTLs recognizing these. The objective of this study was to identify and characterize HLA-A2-restricted CTL epitopes derived from the proteins myeloid cell leukemia-1 (Mcl-1) and cyclin B1. This was done by selecting a number of peptides derived from the two proteins using in silico prediction algorithms. Blood from cancer patients was screened for T cell responses against the peptides in an IFN-¿ ELISPOT assay. In addition, the peptides were analyzed for their HLA-A2 binding affinity using the Assembly assay. Subsequently, CTL clones were established by dendritic cell stimulations and limiting dilution. The clones were tested for cytotoxicity in a 51Cr-release assay.Nine Mcl-1-derived peptides were investigated. One of these, Mcl-1(139), elicited spontaneous responses in cancer patients, and a Mcl-1(139) specific CTL clone was able to lyse ex vivo enriched AML blasts, from an HLA-A2 positive acute myeloid leukemia patient, in an HLA-A2-restricted and peptide specific manner. Twelve cyclin B1-derived peptides were selected. Two of these, CB9(204) and CB10(203), elicited powerful and frequent spontaneous responses in cancer patients. However, CB9(204) also elicited powerful and frequent responses in healthy donors. CB10(203) specific CTL clones were established, and one of these were able to lyse two melanoma cancer cell lines. HLA class I-restriction experiments were, however, not convincing, indicating only partially HLA class I-restriction. Furthermore, two cold target inhibition assays showed differing results, indicating that not a single CTL clone, but at least two clones, were responsible for the observed lysis. Fur

This study describes easy purification methods for mink IgG, IgA and IgM immunoglobulins. IgG and IgM were purified from normal mink serum, while IgA was purified from mink bile from healthy animals. By SDS-polyacrylamid-gel-electrophoresis (SDS-PAGE) and immunoblotting under reducing conditions the estimated molecular weights of the immunoglobulin gamma, alpha and mu heavy chains were found to be 54 kDa, 69 kDa and 83 kDa respectively. The purities of purified IgG, IgM and IgA were estimated by immunoglobulin class specific ELISAs to be more than 90% for IgG and IgM, and more than 80% for IgA.

It is well established that gluten-free diet reduces the incidence of type 1 diabetes mellitus (T1D) in non-obese diabetic (NOD) mice, though the mechanism is not known. However, regulatory T cells (Treg) are likely to play an important role. Also, it is known that dietary gluten induces an intestinal increase in the bacterium Lactococcus garvieae, but the importance of this phenomenon for T1D development is doubtful. Our hypothesis is that gluten is responsible for mediating its effect on T1D through the influence on Treg development independent of gluten-induced Lactococci. Four groups of female NOD and BALB / c mice of 3 week old were fed either a gluten-free diet or a standard diet. Lactococcus garvieae or saline water was administered per oral to one of each dietary group. Spleen and Peyer's patches were sampled from BALB / c mice for flow cytometric monitoring of IL-10 and Treg. NOD mice were diagnosed diabetic with blood glucose level >12 mmol / l. Dietary gluten significantly decreased the occurrence of Tregs by 10-15% (P < 0.05) in mice compared with those fed a standard diet. These results and the diabetes incidence were independent of the gluten-induced bacterial factor Lactococci. The prevalence of Treg was 5- to 10-fold more abundant in the Peyer's patches than in the spleen (P < 0.001). In conclusion, dietary gluten has a significant negative quantitative impact on the generation of Treg in mice, independent of gluten-induced Lactococcus garvieae, and Treg are far more abundant in Peyer's patches than in the spleen.

The capsid of foot-and-mouth disease virus (FMDV) displays several independent B cell epitopes, which stimulate the production of neutralising antibodies. Some of these epitopes are highly variable between virus strains, but dominate the immune response. The site A on VP1 is the most prominent example of a dominant and variable site. This variability is a problem when designing vaccines against this disease, because it necessitates a close match between vaccine strain and virus in an outbreak. We have introduced a series of mutations into viral capsid proteins with the aim of selectively silencing two dominant and highly variable epitopes and thereby divert immune responses toward less dominant but more conserved, protective epitopes. When mice were immunized with modified antigens, the resulting immune responses showed a higher degree of cross-reactivity towards heterologous virus as compared to mice vaccinated with wild type epitopes. Most of the modifications did not adversely affect the ability of the plasmids to induce complete protection of mice against homologous challenge.

The pig is a natural host for Schistosoma japonicum and a useful animal model of human infection. The aim of the present study was to assess the differences between the cytokine profiles in prenatally or postnatally S. japonicum exposed pigs. Seven prenatally exposed pigs, 7 postnatally exposed pigs and 4 uninfected control pigs were compared 27 weeks post exposure. Variables included worm burdens, tissue egg counts, liver pathology and mRNA levels of IL-2, IL-4, IL-10, IL-12, TNF-alpha, TGF-beta 1 and IFN-gamma in the liver and the caecum, assessed by RT-PCR. Infection intensity and level of septal fibrosis were significantly higher in the postnatal group compared to the prenatal group (P <0.05). A significant increase of IL-4 (P <0.01), IL-10 (P <0.01), IL-12 (P <0.01) and TNF-alpha (P <0.05) mRNA level was also observed in the caecum of prenatally infected animals compared to the control group (P <0.01). The prenatal group showed higher levels of TGF-beta 1 in the liver compared with the postnatally infected group (P <0.05) and the control group (P <0.01). This suppressive immune response correlated with previously reported low hepatic pathogenesis in prenatally exposed pigs.