STxB as an Antigen Delivery Tool for Mucosal Vaccination

Abstract

Immunotherapy against cancer and infectious disease holds the promise of high efficacy with minor side effects. Mucosal vaccines to protect against tumors or infections disease agents that affect the upper airways or the lung are still lacking, however. One mucosal vaccine candidate is the B-subunit of Shiga toxin, STxB. In this review, we compare STxB to other immunotherapy vectors. STxB is a non-toxic protein that binds to a glycosylated lipid, termed globotriaosylceramide (Gb3), which is preferentially expressed by dendritic cells. We review the use of STxB for the cross-presentation of tumor or viral antigens in a MHC class I-restricted manner to induce humoral immunity against these antigens in addition to polyfunctional and persistent CD4⁺ and CD8⁺ T lymphocytes capable of protecting against viral infection or tumor growth. Other literature will be summarized that documents a powerful induction of mucosal IgA and resident memory CD8⁺ T cells against mucosal tumors specifically when STxB-antigen conjugates are administered via the nasal route. It will also be pointed out how STxB-based vaccines have been shown in preclinical cancer models to synergize with other therapeutic modalities (immune checkpoint inhibitors, anti-angiogenic therapy, radiotherapy). Finally, we will discuss how molecular aspects such as low immunogenicity, cross-species conservation of Gb3 expression, and lack of toxicity contribute to the competitive positioning of STxB among the different DC targeting approaches. STxB thereby appears as an original and innovative tool for the development of mucosal vaccines in infectious diseases and cancer.

Keywords: GL-Lect; TRM; chemotherapy; cross-presentation; cytotoxic CD8+ T lymphocyte; endosomal escape; glycolipid-lectin; immune checkpoint; radiotherapy; tissue resident memory T cells.

Figure 1

STxB trafficking into cells. Left: Shiga holotoxin molecules are composed of a STxB homopentamer (green) and a catalytic A-subunit (red), which are non-covalently associated. STxB binds to the plasma membrane of target cells via the glycosphingolipid Gb3 (not shown). STxB induces an increment of spontaneous curvature, which upon membrane-mediated clustering of several toxin molecules leads to the formation of endocytic pits from which clathrin-independent carriers are generated for toxin trafficking to early endosomes. From there, the holotoxins are transported via the retrograde trafficking route to the endoplasmic reticulum (ER), via the Golgi apparatus. The catalytic A-subunit is then translocated to the cytosol where it inhibits protein biosynthesis by modifying ribosomal RNAs (not shown). Right: In STxB (green)-based vaccines, antigens (blue) are linked via covalent bonds to the vector. The endocytic process then operates as for Shiga holotoxin molecules. While STxB-antigen conjugates also undergo retrograde trafficking (not shown), a small fraction of them escapes from the lumen of endosomes to reach the cytosol (endosomal escape). Here, proteasomes process the antigens to generate antigenic peptides, that are then imported into the lumen of the ER (or of endo/phagosomal processing compartments; not shown) for loading onto MHC class I molecules and subsequent presentation at the plasma membrane to CD8⁺ T cells.

Figure 2

Optimization of vaccines by delivering antigens to dendritic cells. Different vaccine delivery systems preferentially target antigens to dendritic cells, which are known for their capacity to prime naive T cells: Vectors derived from toxin subunits such as the non-toxic STxB, which binds to the glycosphingolipid Gb3, or adenylate cyclase A, which binds to CD11b; antibodies targeting lectins (DEC-205, DC Sign) or other surface markers (mannose receptor, XCR1, Clec9a) of which some are specifically expressed on DC subpopulations. See text for details.

Figure 3

Vaccine strategies. (a) Chemical coupling. In this procedure, vector and antigen are produced in parallel and then chemically coupled to generate the vaccine. This approach has been extensively used for STxB. (b) Genetic fusion. Vector and antigen are genetically fused at the cDNA level, and then expressed in and purified from prokaryotic or eukaryotic cell systems. In some cases, this approach has been used for STxB, but often corresponding fusion proteins could not be obtained. (c) Nucleic acid vaccines. Fusion proteins between vectors and antigens are expressed from DNA or mRNA molecules that are directly injected into the organism. These vaccine molecules are thereby produced by the cells of the organism receiving the vaccine. The main advantages and disadvantages of the different strategies are listed to the right.