Anne M. Donachie

Biodegradable microparticles for oral immunization

Ovalbumin (OVA) was entrapped in poly(lactide-co-glycolide) microparticles and administered to mice. Following intraperitoneal immunization, the microparticles induced both proliferative T-cell responses and cytotoxic T-cell responses in spleen cells. Following oral immunization, the mean salivary IgA antibody response to microparticles was significantly greater than the response to soluble OVA (p < 0.0001). Serum IgG antibody levels were also significantly greater in the group administered microparticles (p < 0.001). Cholera toxin B subunit was also entrapped in microparticles. Following oral immunization in mice, specific antibody-secreting cells were detected both in the spleens and in the mesenteric lymph nodes.

ISCOMS — a novel strategy for mucosal immunization?

Abstract Orally-active vaccines containing purified or recombinant antigens are highly desirable, particularly in immunization against diseases o f mucosal surfaces, but their development to date has been limited and fitful, hampered by a range o f difficulties. Here, Allan Mowat and Anne Donachie suggest that lipophilic immune-stimulating complexes (ISCOMS) may provide an oral immunization vector for the in duction of a wide range o f immune responses to protein antigens.

CTA1-DD-Immune Stimulating Complexes: a Novel, Rationally Designed Combined Mucosal Vaccine Adjuvant Effective with Nanogram Doses of Antigen

Mucosally active vaccine adjuvants that will prime a full range of local and systemic immune responses against defined antigenic epitopes are much needed. Cholera toxin and lipophilic immune stimulating complexes (ISCOMS) containing Quil A can both act as adjuvants for orally administered Ags, possibly by targeting different APCs. Recently, we have been successful in separating the adjuvant and toxic effects of cholera toxin by constructing a gene fusion protein, CTA1-DD, that combines the enzymatically active CTA1-subunit with a B cell-targeting moiety, D, derived from Staphylococcus aureus protein A. Here we have extended this work by combining CTA1-DD with ISCOMS, which normally target dendritic cells and/or macrophages. ISCOMS containing a fusion protein comprising the OVA323–339 peptide epitope linked to CTA1-DD were highly immunogenic when given in nanogram doses by the s.c., oral, or nasal routes, inducing a wide range of T cell-dependent immune responses. In contrast, ISCOMS containing the enzymatically inactive CTA1-R7K-DD mutant protein were much less effective, indicating that at least part of the activity of the combined vector requires the ADP-ribosylating property of CTA1. No toxicity was observed by any route. To our knowledge, this is the first report on the successful combination of two mechanistically different principles of adjuvant action. We conclude that rationally designed vectors consisting of CTA1-DD and ISCOMS may provide a novel strategy for the generation of potent and safe mucosal vaccines.

Oral vaccination with immune stimulating complexes

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.

The mucosal adjuvant effects of cholera toxin and immune-stimulating complexes differ in their requirement for IL-12, indicating different pathways of action.

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‐γ‐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‐β levels, suggesting that the immunomodulating effect of CT was independent of IL‐12 as well as TGF‐β 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.

Immune stimulating complexes as mucosal vaccines

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.

The Combined CTA1-DD/ISCOM Adjuvant Vector Promotes Priming of Mucosal and Systemic Immunity to Incorporated Antigens by Specific Targeting of B Cells

The cholera toxin A1 (CTA1)-DD/QuilA-containing, immune-stimulating complex (ISCOM) vector is a rationally designed mucosal adjuvant that greatly potentiates humoral and cellular immune responses. It was developed to incorporate the distinctive properties of either adjuvant alone in a combination that exerted additive enhancing effects on mucosal immune responses. In this study we demonstrate that CTA1-DD and an unrelated Ag can be incorporated together into the ISCOM, resulting in greatly augmented immunogenicity of the Ag. To demonstrate its relevance for protection against infectious diseases, we tested the vector incorporating PR8 Ag from the influenza virus. After intranasal immunization we found that the immunogenicity of the PR8 proteins were significantly augmented by a mechanism that was enzyme dependent, because the presence of the enzymatically inactive CTA1R7K-DD mutant largely failed to enhance the response over that seen with ISCOMs alone. The combined vector was a highly effective enhancer of a broad range of immune responses, including specific serum Abs and balanced Th1 and Th2 CD4+ T cell priming as well as a strong mucosal IgA response. Unlike unmodified ISCOMs, Ag incorporated into the combined vector could be presented by B cells in vitro and in vivo as well as by dendritic cells; it also accumulated in B cell follicles of draining lymph nodes when given s.c. and stimulated much enhanced germinal center reactions. Strikingly, the enhanced adjuvant activity of the combined vector was absent in B cell-deficient mice, supporting the idea that B cells are important for the adjuvant effects of the combined CTA1-DD/ISCOM vector.

The role of antigen‐presenting cells and interleukin‐12 in the priming of antigen‐specific CD4+ T cells by immune stimulating complexes

Immune stimulating complexes (ISCOMs) containing the saponin adjuvant Quil A are vaccine adjuvants that promote a wide range of immune responses in vivo, including delayed‐type hypersensitivity (DTH) and the secretion of both T helper 1 (Th1) and Th2 cytokines. However, the antigen‐presenting cell (APC) responsible for the induction of these responses has not been characterized. Here we have investigated the role of dendritic cells (DC), macrophages (Mφ) and B cells in the priming of antigen‐specific CD4+ T cells in vitro by ISCOMs containing ovalbumin (OVA). OVA ISCOMs pulsed bone marrow (BM)‐derived DC but not BM Mφ, nor naïve B cells prime resting antigen‐specific CD4+ T cells, and this response is greatly enhanced if DC are activated with lipopolysaccharide (LPS). Of the APC found in the spleen, only DC had the capacity to prime resting antigen specific CD4+ T cells following exposure to OVA ISCOMs in vitro, while Mφ and B cells were ineffective. DC, but not B cells purified from the draining lymph nodes of mice immunized with OVA ISCOMs also primed resting antigen‐specific CD4+ T cells in vitro, suggesting that DC are also critical in vivo. Using DC and T cells from interleukin (IL)‐12 p40−/− mice, we also identified a crucial role for IL‐12 in the priming of optimal CD4+ T cell responses by OVA ISCOMs. We suggest that DC are the principal APC responsible for the priming of CD4+ T cells by ISCOMs in vivo and that directed targeting of these vectors to DC may enhance their efficancy as vaccine adjuvants.

Simultaneous presentation and cross-presentation of immune-stimulating complex-associated cognate antigen by antigen-specific B cells

We demonstrate that uptake of oligomeric cognate antigen (OVA‐hen egg lysozyme, OVA‐HEL) alone or incorporated in immune‐stimulating complexes (ISCOMS) facilitates presentation and simultaneous cross‐presentation of OVA by HEL‐specific B cells in vitro. HEL‐specific B cells stimulated CD8+ T cell responses in vitro to the same extent as bone marrow‐derived dendritic cells. Cross‐presentation by specific B cells required endosomal acidification, proteasomal processing and classical MHC class I/peptide transport. Specific B cells also acquired both antigens rapidly in vivo and presented them to CD4+ T cells. However, only HEL‐specific B cells from OVA‐HEL ISCOMS‐immunised mice could cross‐present OVA to naive OVA‐specific CD8+ T cells. Antigen‐specific B cells were also activated selectively by OVA‐HEL ISCOMS in vitro and importantly, the presence of HEL‐specific B cells promoted the persistence of clonal expansion of OVA‐specific CD8+ T cells after in vivo immunisation with OVA‐HEL ISCOMS. These results demonstrate preferential MHC class I and class II processing of cognate antigen incorporated in ISCOMS by specific B cells in vitro and in vivo, highlighting the ability of ISCOMS to target B cells and offering novel insights into the role of B cells in cross‐presentation to CD8+ T cells.

Dendritic cell maturation enhances CD8+ T-cell responses to exogenous antigen via a proteasome-independent mechanism of major histocompatibility complex class I loading

Immune stimulating complexes (ISCOMS) containing the saponin adjuvant Quil A are vaccine adjuvants that induce a wide range of immune responses in vivo, including strong class I major histocompatibility complex (MHC) ‐restricted cytotoxic T‐lymphocyte activity. However, the antigen‐presenting cell responsible for the induction of these responses has not been characterized. Here we have investigated the role of dendritic cells (DC) in the priming of antigen‐specific CD8+ T cells in vitro by ISCOMS containing ovalbumin. Resting bone marrow DC pulsed with ovalbumin ISCOMS efficiently prime resting CD8+ T cells through a mechanism that is transporter associated with antigen processing (TAP) dependent, but independent of CD40 ligation and CD4+ T‐cell help. Lipopolysaccharide‐induced maturation of DC markedly enhances their ability to prime CD8+ T cells through a mechanism which is also independent of CD4+ T‐cell help, but is dependent on CD40 ligation. Furthermore, DC maturation revealed a TAP‐independent mechanism of CD8+ T‐cell priming. Our results also show that class I MHC‐restricted presentation of ovalbumin in ISCOMS by DC is sensitive to chloroquine and brefeldin A but insensitive to lactacystin. We suggest that DC may be the principal antigen‐presenting cells responsible for the priming of CD8+ T cells by ISCOMS in vivo and that targeting these vectors to activated DC may enhance their presentation via a novel pathway of class I antigen processing.