Inverse vaccines that tolerogenically target antigens to antigen-presenting cells (APCs) offer promise in prevention of immunity to allergens and protein drugs and treatment of autoimmunity. We have previously shown that targeting hepatic APCs through intravenous injection of synthetically glycosylated antigen leads to effective induction of antigen-specific immunological tolerance. Here, we demonstrate that targeting these glycoconjugates to lymph node (LN) APCs under homeostatic conditions leads to local and increased accumulation in the LNs compared to unmodified antigen and induces a tolerogenic state both locally and systemically. Subcutaneous administration directs the polymeric glycoconjugate to the draining LN, where the glycoconjugated antigen generates robust antigen-specific CD4+ and CD8+ T cell tolerance and hypo-responsiveness to antigenic challenge via a number of mechanisms, including clonal deletion, anergy of activated T cells, and expansion of regulatory T cells. Lag-3 up-regulation on CD4+ and CD8+ T cells represents an essential mechanism of suppression. Additionally, presentation of antigen released from the glycoconjugate to naïve T cells is mediated mainly by LN-resident CD8+ and CD11b+ dendritic cells. Thus, here we demonstrate that antigen targeting via synthetic glycosylation to impart affinity for APC scavenger receptors generates tolerance when LN dendritic cells are the cellular target.

The only FDA-approved oral immunotherapy for a food allergy provides protection against accidental exposure to peanuts. However, this therapy often causes discomfort or side effects and requires long-term commitment. Better preventive and therapeutic solutions are urgently needed. We have developed an inverse vaccine technology that utilizes glycopolymer-conjugated antigens to induce antigen-specific non-responsiveness. The glycopolymer conjugates were administered intravenously (i.v.) or subcutaneously (s.c.) and were found to traffic to the liver or lymph nodes, respectively, leading to preferential internalization by antigen-presenting cells, educating the immune system to respond in an innocuous way. In a mouse model of cow's milk allergy, treatment with glycopolymer-conjugated β-lactoglobulin (BLG) was effective in preventing the onset of allergy. In addition, s.c. administration of glycopolymer-conjugated BLG showed superior safety and potential in treating existing allergies in combination with an anti-CD20 co-therapy. This platform may provide an antigen-specific immunomodulatory strategy to prevent and treat food allergies.

The only FDA-approved oral immunotherapy for a food allergy provides protection against accidental exposure to peanuts. However, this therapy often causes discomfort or side effects and requires long-term commitment. Better preventive and therapeutic solutions are urgently needed. We have developed a technology for inverse vaccination using glyco-polymerized antigens to induce antigen-specific non-responsiveness. We have demonstrated that following subcutaneous (SC) administration, glyco-polymer conjugates traffic to draining lymph nodes (LNs) and are preferentially internalized by antigen presenting cells, educating the immune system to respond to these antigens in an innocuous way. Here, we tested SC administration of glyco-polymerized β-lactoglobulin (BLG) in a murine model of cow’s milk allergy. Two doses of glyco-polymerized BLG given one week apart (prior to sensitization) prevented an allergic response to BLG upon intragastric challenge as measured by reductions in BLG-specific IgG, IgG1, and IgE production as well as BLG-specific type 2 T helper cell (Th2) responses in vitro. We further explored the therapeutic potential of glyco-polymerized BLG. Unlike unmodified BLG, two SC injections of glyco-polymerized BLG after sensitization did not cause anaphylactic reactions in allergic mice and inhibited cellular Th2 cytokines upon BLG restimulation. Since pre-existing humoral immunity inhibited the therapeutic effect of our glyco-polymerized BLG, we introduced a co-therapy (anti-CD20) to blunt the humoral response and observed ameliorated anaphylactic responses following glyco-polymerized BLG therapy. This platform may provide a potential T cell-modulating strategy to prevent and treat food allergies. This work was supported in part by seed funding from the Chicago Immunoengineering Innovation Center at the University of Chicago as well as the Food Allergy Fund.

Lymphatic endothelial cells (LECs) have been shown to archive antigen (Ag), i.e. retain Ag in its native form, for weeks after exposure. We have developed a polymeric platform that promotes targeting of Ag to lymph node (LN)-resident LECs by exploiting the sugar-binding scavenger receptors expressed by LECs, leading to longer Ag retention over 2 weeks. This targeting results in enhanced Ag archiving in LECs, even in the absence of an adjuvant or other inflammatory stimuli previously shown to be necessary for Ag archiving in LECs. 2 weeks after subcutaneous (s.c.) delivery of unmodified OVA, we observed substantial proliferation and activation of adoptively transferred OVA-specific CD8+ T cells. Immunization and subsequent Ag archiving with our targeting polymer conjugated to OVA (without adjuvant) led to reduced CD8+ T cell proliferation but higher differentiation into a central memory phenotype, both by surface receptors as well as the ratio of EOMES vs. T-bet on activated T cells. We measured MHCI-mediated presentation of OVA (SIINFEKL presented on the H-2kb molecule) on LN-LECs vs. DCs over two weeks following s.c. injection and found that Ag presentation by LECs was comparable to DCs. This demonstrates that LECs are able to cross-present archived Ag directly to CD8+ T cells, with potential implications in the prolonged maintenance of memory CD8+ T cells and design of a more effective vaccine platform.