Vitamin D3 modulates the function of chicken macrophages.

Vitamin D3 is known to modulate both innate and adaptive immune responses in mammals, but there is little information on its effects on avian immune system cells. Here, we studied the effects of vitamin D3 on chicken macrophages. Chicken macrophages expressed vitamin D receptor (VDR) and lipopolysaccharide (LPS) stimulation increased their VDR expression. Macrophages were treated with 1,25(OH)2D3 in the presence or absence of Toll-like receptor ligands, such as LPS and Pam3CSK4. Subsequently, macrophage activation was assessed by measuring nitric oxide (NO) and expression of CXCL8 and interleukin (IL)-1ß. In addition, changes in major histocompatibility complex (MHC)-II and CD86 were examined. Treatment of cells with 1,25(OH)2D3 increased the ability of macrophages to respond to stimuli and produce NO, but vitamin D3 alone did not activate macrophages and resulted in the down-regulation of CD86, MHC-II, CXCL8 and IL-1ß. These findings suggest that vitamin D3 has an immunomodulatory role in chicken macrophages.

Expansion of anti-AFP Th1 and Tc1 responses in hepatocellular carcinoma occur in different stages of disease.

BACKGROUND: alpha-Fetoprotein (AFP) is a tumour-associated antigen in hepatocellular carcinoma (HCC) and is a target for immunotherapy. However, there is little information on the pattern of CD4 (Th1) and CD8 (Tc1) T-cell response to AFP in patients with HCC and their association with the clinical characteristics of patients. METHODS: We therefore analysed CD4 and CD8 T-cell responses to a panel of AFP-derived peptides in a total of 31 HCC patients and 14 controls, using an intracellular cytokine assay for IFN-gamma. RESULTS: Anti-AFP Tc1 responses were detected in 28.5% of controls, as well as in 25% of HCC patients with Okuda I (early tumour stage) and in 31.6% of HCC patients with stage II or III (late tumour stages). An anti-AFP Th1 response was detected only in HCC patients (58.3% with Okuda stage I tumours and 15.8% with Okuda stage II or III tumours). Anti-AFP Th1 response was mainly detected in HCC patients who had normal or mildly elevated serum AFP concentrations (P=0.00188), whereas there was no significant difference between serum AFP concentrations in these patients and the presence of an anti-AFP Tc1 response. A Th1 response was detected in 44% of HCC patients with a Child-Pugh A score (early stage of cirrhosis), whereas this was detected in only 15% with a B or C score (late-stage cirrhosis). In contrast, a Tc1 response was detected in 17% of HCC patients with a Child-Pugh A score and in 46% with a B or C score. CONCLUSION: These results suggest that anti-AFP Th1 responses are more likely to be present in patients who are in an early stage of disease (for both tumour stage and liver cirrhosis), whereas anti-AFP Tc1 responses are more likely to be present in patients with late-stage liver cirrhosis. Therefore, these data provide valuable information for the design of vaccination strategies against HCC.

The effects of DNA containing CpG motif on dendritic cells.

Dendritic cells (DC) are specialized antigen-presenting cells. DC can acquire and process antigens in the periphery before maturing and migrating to secondary lymphoid tissues where they present the antigens and deliver co-stimulatory signals to T cells. We describe an immunostimulatory oligonucleotide containing a CpG motif that stimulated murine DC to up-regulate co-stimulatory molecules, induce T-cell proliferative responses and secrete interleukin-12 in vitro. Administration of this oligonucleotide, but not of a control oligonucleotide lacking this motif, to mice led to the disappearance of DC from the marginal zone and T-cell areas of spleen, but not from heart or kidney. The same CpG did not cause maturation of monocyte-derived human DC in vitro, but lipopolysaccharide-treated monocyte-derived DC showed enhanced functional activity and up-regulated co-stimulatory molecules.

Quillaja saponin formulations that stimulate proinflammatory cytokines elicit a potent acquired cell-mediated immunity.

We examined the ability of various Quillaja saponins in iscom-matrix formulations to induce proinflammatory cytokines, such as interleukin (IL)-1alpha and IL-6, and to stimulate acquired immune responses to influenza virus envelope proteins. The A-fraction of Quillaja saponins (QH-A) was shown to stimulate antigen-presenting cells (APC) to produce proinflammatory cytokines, and elicited a high primary antigen-specific antibody response and potent cell-mediated responses, as measured by T-cell proliferation, production of cytokines and cytotoxic T-lymphocyte (CTL) activity. The C-fraction of Quillaja saponins (QH-C) was shown to have a low capacity to stimulate proinflammatory cytokines and elicited low primary antibody and T-cell responses. However, the QH-C iscom-matrix mediated a potent booster effect, resulting in a high secondary antibody response. The ability of APC to discriminate and to respond to QH-A formulations more efficiently than to QH-C with release of proinflammatory cytokines, which precedes a potent acquired immune response, identifies an important mechanism through which some adjuvants may exert their immunoenhancing activities.

Internalization of iscom-borne antigens and presentation under MHC class I or class II restriction.

Exogenous nonreplicating antigens (Ag) incorporated into immunostimulating complexes (iscoms) induce CTL responses under MHC class I restriction. A requirement for inducing CTL responses is that the Ag is delivered to the cytosol of antigen-presenting cells (APC), a route restricted to endogenously produced Ag. To investigate the mechanisms by which iscoms elicit MHC class I-restricted responses, the intracellular distribution of influenza virus envelope proteins incorporated in iscoms (flu-iscoms) or in micelles (flumicelles) was studied in vitro using murine peritoneal cells (PEC). Ultrathin sections of cells pulsed with biotinylated flu-iscoms or flu-micelles were analyzed by electron microscopy after detection of the biotin label by reaction with streptavidin-gold. PEC pulsed with flu-iscoms showed a pattern of scattered gold particles distributed in clear and dense vesicles as well as in the intracellular space but not associated with organelles. In cells pulsed with flu-micelles, Ag was also detected in most cellular compartments but at a considerably lower concentration. The intracellular distribution of particulate Ag in iscom or micelle form was confirmed by lysis and differential centrifugation of Ag-pulsed APC. Furthermore, P815 cells pulsed with flu-iscoms were lysed by specific immune effectors showing that the iscom-Ag was processed and presented by class I-expressing APC. Flu-iscoms were internalized about 50-fold more efficiently than ovalbumin iscoms (ovaiscoms) suggesting that the nature of the protein and/or the presence of cellular receptors are important factors influencing the capacity of APC to take up iscom-borne proteins. PEC accounted for the most active internalization of iscom-borne Ag, although splenic dendritic cells and B cells also took up fluiscoms with remarkable efficiency.

Effects of carbohydrate modification of Quillaja saponaria Molina QH-B fraction on adjuvant activity, cholesterol-binding capacity and toxicity.

The iscom is an efficient antigen-presenting system for various antigens inducing both MHC class I and class II restricted immune responses. Protective immunity has been evoked against a variety of infectious agents. The saponin adjuvant Quil A, which was originally used to form iscoms, is composed of a mixture of structurally similar triterpenoids from Quillaja saponaria Molina having different biological activities. A purified, toxic Quillaja triterpenoid fraction with strong adjuvant activity, designated QH-B, was used to study whether modification of the carbohydrate moiety with sodium periodate would alter the toxicity without harming adjuvant activity and cholesterol-binding capacity. Most sugars, and in particular Api, Gal and Xyl, were modified by periodate treatment with only minor changes of the molecular weights indicating no loss of sugar residues. The adjuvant activity of QH-B was reduced in a dose-related manner, and at a concentration of 25 mM sodium periodate a significant reduction in toxicity was observed. The differences in both toxicity and adjuvant activity of the periodate-treated QH-B could be derived from alterations in the structure of the sugars Gal and Xyl, while modification of Api may influence adjuvant activity but not toxicity in vivo. The cholesterol-binding capacity, a prerequisite for iscom formation, was not affected by periodate oxidation at the doses tested. However, the use of modified QH-B as described in the present study for iscom-matrix formation resulted in "saponin-lipid complexes" which, to a various degree or totally, deviated from the characteristic iscom morphology.

In vivo and in vitro induction of IL-6 by Quillaja saponaria molina triterpenoid formulations.

Quillaja saponaria Molina and some of the defined Quillaja components are potent adjuvants. An important function of adjuvants is the activation of antigen-presenting cells (APC), a prerequisite for the development of immune responses. Interleukin 6 (IL-6) has been identified as a key factor in the generation of cytolytic T lymphocytes, which constitute an important effector mechanism elicited by immuno-stimulatory complex (iscom)-borne antigens. To identify factors relevant to the unique property of iscoms to mediate CTL responses, we analysed the capacity of different defined Quillaja triterpenoid components in various formulations to stimulate production of IL-6 by APC in vitro and in vivo. The iscom formed with Quillaja adjuvant and incorporated influenza virus envelope proteins elicited the highest secretion of IL-6. The production of IL-6 was also stimulated by the antigen free matrix of the iscom and even by the Quillaja triterpenoids as free components albeit to a significantly lesser extent. Among the various combinations of QH-A and QH-C tested and also the original semipurified spikoside, the QH 7.0.3 matrix was the most efficient formulation for activation of IL-6 production by APC. In general, an increasing proportion of QH-A vs QH-C increases the capacity to activate APC. The results demonstrate that the incorporated antigen and the adjuvant component in the same particle have the synergistic effects on immunogenicity.

Immunomodulation by Quillaja saponaria adjuvant formulations: in vivo stimulation of interleukin 12 and its effects on the antibody response.

The capacity of adjuvants to activate Ag-presenting cells during the induction of the primary immune response is of critical importance for the development of protective immunity to a number of pathogens. In this context, interleukin 12 (IL-12) has a key role by controlling the differentiation of T helper cells and favouring the expansion of Th1 cells. The capacity of iscoms with influenza virus Ag (flu-iscoms) and iscom matrix with EBV gp340 Ag to induce IL-12 was analysed in mice. The flu-iscom drives the immune response towards a Th1 type subsequent to IL-12 induction as measured in the serum of H2b, H2d and H2k mice. The iscom presenting the Ag and adjuvant in the same particle was considerably more efficient than the formulation of matrix and Ag in separate particles. Inhibition experiments with mAb neutralizing IL-12, interferon gamma (IFN-gamma) or IL-4, the latter two cytokines representing the Th1 and Th2 type of responses, showed that iscoms induce a broader immune response than that involving IL-12. This was shown by the additional effect that IL-4 neutralization had on the immune response to iscoms. Anti-IL 12 reduced the specific total Ab as well as IgG1, IgG2a and IgG2b while anti-IL 4 influenced the response to iscom by decreasing IgG2a and increasing IgG1. Further, the neutralization experiments indicate that IL-12 has a broader effect than IFN-gamma on the Ab response by influencing the production of IgG1, IgG2a and IgG2b.

In vitro activation of antigen-presenting cells (APC) by defined composition of Quillaja saponaria Molina triterpenoids.

The capacity of adjuvants to stimulate cytokine production by APC is important for the initiation of the immune response. Novel adjuvant formulations based on the iscom technology have been developed using selected triterpenoid components from Quillaja saponaria Molina. Five of these new Quillaja formulations were used to prepare matrix (an antigen-free particle) and tested for their capacity to stimulate IL-1 secretion by murine peritoneal cells in vitro. The formulation denominated QH 7.0.3 was superior to the other matrix formulations, including the original spikoside matrix. The QH 7.0.3 formulation in iscoms containing influenza virus envelope antigens induced IL-1 secretion more efficiently than the antigen-free matrix, or a mixture of matrix and viral antigens, or the free Quillaja components of similar composition. Compared with adjuvants known as IL-1 inducers, QH 7.0.3 flu-iscoms were as efficient as the most prominent IL-1 inducer, i.e. lipopolysaccharide (LPS) and superior to cholera toxin (CT) and muramyl dipeptide (MDP). These results indicate that the composition per se of triterpenoids included in iscoms or matrix has a prominent influence on the level of APC activation which may result in qualitatively different immune responses in vivo.

Isolation and quantification of Quillaja saponaria Molina saponins and lipids in iscom-matrix and iscoms.

In the iscom, multiple copies of antigen are attached by hydrophobic interaction to a matrix which is built up by Quillaja triterpenoid saponins and lipids. Thus, the iscom presents antigen in multimeric form in a small particle with a built-in adjuvant resulting in a highly immunogenic antigen formulation. We have designed a chloroform-methanol-water extraction procedure to isolate the triterpenoid saponins and lipids incorporated into iscom-matrix and iscoms. The triterpenoids in the triterpenoid phase were quantitated using orcinol sulfuric acid detecting their carbohydrate chains and by HPLC. The cholesterol and phosphatidylcholine in the lipid phase were quantitated by HPLC and a commercial colorimetric method for the cholesterol. The quantitative methods showed an almost total separation and recovery of triterpenoids and lipids in their respective phases, while protein was detected in all phases after extraction. The protein content was determined by the method of Lowry and by amino acid analysis. Amino acid analysis was shown to be the reliable method of the two to quantitate proteins in iscoms. In conclusion, simple, reproducible and efficient procedures have been designed to isolate and quantitate the triterpenoids and lipids added for preparation of iscom-matrix and iscoms. The procedures described should also be useful to adequately define constituents in prospective vaccines.