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There is a need for non-living adjuvant vectors that will allow a full range of local and systemic immune responses to orally administered purified antigens. Here we describe our experience with lipophilic immune-stimulating complexes (ISCOMs) containing the saponin adjuvant Quil A. When given orally, ISCOMs containing the model protein antigen ovalbumin (OVA) induce a wide range of systemic immune responses, including Th1 and Th2 CD4-dependent activity, serum IgG antibodies and class I MHC-restricted cytotoxic T cell responses. In addition, there is local production of secretory IgA antibodies in the intestine itself, as well as priming of CD4 and CD8 T cell responses in the draining lymphoid tissues. Preliminary results indicate that the mucosal adjuvant properties of ISCOMs may reflect their ability to deliver antigen combined with the pro-inflammatory properties of Quil A in a particulate form. Of the many inflammatory mediators induced, interleukin-12, derived from dendritic cells and/or macrophages, appears to be of central importance. These results indicate that ISCOMs may prove to be useful mucosal vaccine vectors with functions which are distinct from existing vectors of this type.

MyD-1 (CD172) is a member of the family of signal regulatory phosphatase (SIRP) binding proteins, which is expressed on human CD14+ monocytes and dendritic cells. We now show a novel role for MyD-1 in the regulation of the innate immune system by pathogen products such as lipopolysaccharide (LPS), purified protein derivative (PPD), and Zymosan. Specifically, we demonstrate that ligation of MyD-1 on peripheral blood mononuclear cells (PBMCs) inhibits tumor necrosis factor alpha (TNFα) secretion but has no effect on other cytokines induced in response to each of these products. In an attempt to understand the molecular mechanisms underlying this surprisingly selective effect we investigated signal transduction pathways coupled to MyD-1. Ligation of the SIRP was found to recruit the tyrosine phosphatase SHP-2 and promote sequential activation of phosphatidylinositol (PI) 3-kinase, phospholipase D, and sphingosine kinase. Inhibition of LPS-induced TNFα secretion by MyD-1 appears to be mediated by this pathway, as the PI 3-kinase inhibitor wortmannin restores normal LPS-driven TNFα secretion. MyD-1-coupling to this PI 3-kinase-dependent signaling pathway may therefore present a novel target for the development of therapeutic strategies for combating TNFα production and consequent inflammatory disease. (Blood. 2003;102:2532-2540)

Signal inhibitory regulatory proteins are a family of transmembrane glycoproteins involved in the negative regulation of receptor tyrosine kinase signaling pathways. MyD-1 is a recently described member of this family. In this report we have assessed the function of MyD-1 in regulating T cell function utilizing an anti-MyD-1-specific monoclonal antibody (mAb). We show that ligating MyD-1 on antigen-presenting cells (APC) inhibits subsequent T cell activation induced by either anti-CD3 mAb or by allogeneic APC but has no effect on responses to either tuberculin purified protein derivative or tetanus toxoid antigens. Moreover, we show that the inhibition of T cell responses is not due to any change of costimulatory molecule expression on APC. We observed that the production of TNFalpha, a cytokine that we have earlier identified as important in the mechanism of MyD-1 immune regulation, is inhibited by cross-linking of MyD-1. We further show that TNFalpha is critically important in the regulation of T cell responses in the absence of danger signals, and indeed addition of TNFalpha can overcome the inhibitory effects of anti-MyD-1 antibody. This information may lead to a better understanding of the regulation of T cell responses in the absence of danger and therefore offer a possible therapeutic target to modulate aberrant immune responses.

Lipophilic immune stimulating complexes (ISCOMS) containing the Quil A adjuvant can induce local and systemic immune responses against orally delivered protein antigens. Here, we have examined the possibility that ISCOMS act by modulating local innate immune responses and antigen uptake in the intestine. Feeding ISCOMS to mice induced recruitment of dendritic cells (DCs), activated macrophages (mφ) and lymphocytes into the mesenteric lymph node (MLN), as well as recruitment of macrophages and B cells into the PP. Feeding ISCOMS also enhanced the absorption into circulation of a test feed of ovalbumin (OVA), with accelerated kinetics and increased peak levels. Serum taken 1 h after feeding a tolerogenic dose of OVA to mice given ISCOMS orally did not induce the tolerance of systemic delayed type hypersensitivity (DTH) and antibody responses found in mice receiving serum from donors fed OVA alone. Thus, ISCOMS may increase the immunogenicity of intestinal antigen via a combination of effects on antigen uptake and on local accessory cells.

Adjuvants that can improve mucosal vaccine efficacy are much warranted. In this comparative study between cholera toxin (CT) and immune-stimulating complexes (ISCOM) we found that, contrary to CT, ovalbumin (OVA)-ISCOM were poor inducers of mucosal anti-OVA IgA responses, but induced similar or better systemic immunity following oral immunizations. The addition of CT to the oral OVA-ISCOM protocol did not stimulate local anti-OVA IgA immunity, nor did it change the quality or magnitude of the systemic responses. Both vectors recruited strong innate immunity, but only OVA-ISCOM could directly induce IL-12, demonstrable at the protein and mRNA levels. CT had no inhibitory effects on lipopolysaccharide/IFN-gamma-induced IL-12 mRNA expression or IL-12 production. Furthermore, adjuvanticity of CT was unaffected in IL-12-deficient mice, while OVA-ISCOM showed partly impaired adjuvant effects by the lack of IL-12. CT abrogated the induction of oral tolerance stimulated by antigen feeding in these mice. In addition, CT did not alter TGF-beta levels, suggesting that the immunomodulating effect of CT was independent of IL-12 as well as TGF-beta production. Taken together, these findings indicate that mucosal adjuvanticity of CT and ISCOM are differently dependent on IL-12, suggesting that separate and distinct antigen-processing pathways are involved.

There is a need for non-living adjuvant vectors which will induce a full range of local and systemic immune responses to orally administered purified antigens. Here we describe our experience with lipophilic immune stimulating complexes (ISCOMS) containing the saponin adjuvant Quil A. When given orally, ISCOMS containing the model protein antigen ovalbumin (OVA) induce a wide range of systemic immune responses, including Th1 and Th2 CD4 dependent activity, class I MHC restricted cytotoxic T-cell responses and local production of secretory IgA antibodies. More recent results indicate that ISCOMS may act partly by enhancing the uptake of protein from the gut. In addition, intraperitoneal injection of ISCOMS recruits and activates many components of the innate immune system. including neutrophils, macrophages, and dendritic cells. In parallel, there is increased production of nitric oxide (NO), reactive oxygen intermediates (ROI), interleukins (IL) 1, 6, 12, and gamma interferon (gammaIFN). Of these factors, only IL12 is essential for the immunogenicity of ISCOMS in vivo, as mucosal and systemic responses to ISCOMS are reduced in IL12KO mice, but not in IL4KO, IL6KO, inducible NO synthase (iNOS) KO, or gammaIFN receptor KO mice. We propose that ISCOMS act by targetting antigen and adjuvant to macrophages and/or dendritic cells. This pathway may be amenable to exploitation for vaccine development, especially if combined with another vector with a different mucosal adjuvant profile, such as cholera toxin.

There is currently a great deal of interest in developing orally active synthetic vaccines containing recombinant proteins as protective antigens. However, purified proteins are poorly immunogenic in general and do not elicit the mucosal immunity essential for protecting against diseases of mucosal surfaces such as the intestine and respiratory tract (McGhee et al 1992). Indeed, the usual result of administering soluble proteins by the oral route is profound immunological tolerance which prevents systemic and local immune responses on subsequent exposure to the antigen (Mowat 1987). Thus there is a need for vectors that will avoid this phenomenon of oral tolerance and prime a full range of immune responses to orally administered protein antigens.

Signal inhibitory regulatory proteins are a family of transmembrane glycoproteins involved in the negative regulation of receptor tyrosine kinase signaling pathways. MyD-1 is a recently described member of this family. In this report we have assessed the function of MyD-1 in regulating T cell function utilizing an anti-MyD-1-specific monoclonal antibody (mAb). We show that ligating MyD-1 on antigen-presenting cells (APC) inhibits subsequent T cell activation induced by either anti-CD3 mAb or by allogeneic APC but has no effect on responses to either tuberculin purified protein derivative or tetanus toxoid antigens. Moreover, we show that the inhibition of T cell responses is not due to any change of costimulatory molecule expression on APC. We observed that the production of TNFalpha, a cytokine that we have earlier identified as important in the mechanism of MyD-1 immune regulation, is inhibited by cross-linking of MyD-1. We further show that TNFalpha is critically important in the regulation of T cell responses in the absence of danger signals, and indeed addition of TNFalpha can overcome the inhibitory effects of anti-MyD-1 antibody. This information may lead to a better understanding of the regulation of T cell responses in the absence of danger and therefore offer a possible therapeutic target to modulate aberrant immune responses.