Immunological Response from an Entirely Carbohydrate Antigen: Design of Synthetic Vaccines Based on Tn-PS A1 Conjugates Ravindra A. De Silva, Qianli Wang, Tristan Chidley, Dananjaya K. Appulage, and Peter R. Andreana* Wayne State UniVersity, Department of Chemistry, 5101 Cass AVenue, Detroit, Michigan 48202 Received April 1, 2009; E-mail: pra@chem.wayne.edu The introduction of vaccines into medical practice has been one of the most significant advancements of modern medicine. 1 Its success can be attributed to the fact that vaccinology has expanded beyond its traditional mainstays (attenuated or dead microorganisms, inactivated bacterial toxins, and protein subunit vaccines) to the likes of recom- binant proteins and glycoproteins, synthetic peptides, and conjugate vaccines. 2 Despite the enormous success, there remains a need for effective vaccines for the treatment of serious diseases such as malaria, AIDS, antibiotic-resistant infections, and cancer. Carbohydrates are involved in a wide variety of biological roles. 3 For example, oncogenic transformation of cells is closely correlated with dramatic changes in their glycosylation patterns. 4 Aberrant polysaccharides expressed on these cancer-cell surfaces [so-called tumor-associated carbohydrate antigens (TACAs)] have been used successfully in the diagnosis and prognosis of cancer for many years. 4b Furthermore, TACAs can induce changes in antigenicity and immu- nogenicity in cancer cells, which may allow TACA-derived cancer vaccines and cancer immunotherapies to be realized. 4c Although isolated/pure carbohydrates/polysaccharides have long been known to be T-cell-independent and exhibit poor immunogenic- ity, 5 glycoconjugate vaccines, in which carbohydrate antigens are tethered to strong immunogenic carrier proteins, such as bovine serum albumin (BSA), tetanus toxoid, keyhole limpet hemocyanin, and others, can elicit an MHCII T-cell response characterized by immunoglobulin G (IgG) production. 6 Extensive research has focused on the creation of synthetic vaccines 7 based on complex carbohydrate antigens/epitopes conjugated to these carriers. Such vaccines have shown varying degrees of promise, and as an outcome, the components necessary for eliciting protective antibody production have become more defined. 8 Paradoxi- cally, these conjugate vaccines can be less intrinsically immunogenic, even as they become safer and more precisely targeted. This dichotomy typically arises from nonrelated binding and a strong immune response against the carrier protein, resulting in suppression of carbohydrate- specific antibody production. 9 Also, TACAs are non-site-specifically coupled to carrier proteins, leading to vaccine heterogeneity. Random coupling may also result in modification of important recognition epitomes on the carrier protein, 10 and conjugation chemistry is often difficult to control, leading to glycoproteins with ambiguities in struc- ture and composition, which may affect the reproducibility of an immune response. A recent report has provided convincing evidence for cases in which zwitterionic polysaccharides (ZPSs) invoke an MHCII- mediated T-cell response in the absence of proteins. 11 Kasper and co-workers 5b,12 have identified capsular polysaccharide structures that induce a CD4+ T-cell response known to modulate bacterial abscess formation. An unusual ZPS, PS A1 (1), consisting of a tetrasaccharide-core repeating unit (∼120 units) carrying an elec- trostatic charge character on adjacent monosaccharides, elicits an immune response similar to that for exogenous proteins. In an attempt to overcome some of the current challenges with protein-carbohydrate vaccine development and examine a new direction, we elected to focus our attention on a strategy that would profit from the inherent MHCII-mediated immune activation by 1 and conjugate a known TACA, Tn hapten (3), in order to probe the immunogenicity and specificity for the development of a novel cancer immunotherapy. We sought to exploit an entirely carbohydrate immunogen to potentially overcome nonrelated peptide binding (cross reactivity with “self” proteins), generate carbohydrate-specific antibody production, and capitalize on a complete donor-acceptor sequence of carbohydrates for increased antibody-carbohydrate binding. Our strategy also incorporates a site-specific link between 1 and 3, negating ambiguities in structure and composition. To garner appreciable amounts (∼500 mg) of 1, a large-scale fermentation protocol employing the anaerobe Bacterioides fragilis NTCT 9343 was carried out. 13 Purified 1, devoid of any protein and lipopolysaccharide, was subjected to selective oxidative cleav- age conditions using 0.5 equiv of NaIO 4 in 0.10 M acetate buffer (pH 5.0) (Scheme 1). The oxidation protocol took advantage of the vicinal hydroxyl relationship (1° 6-OH, 2° 5-OH) 12 of the D-galactofuranose motif. The oxidation led to (hydrated) aldehyde 2, providing a chemoselective handle. The addition of 5 equiv of KCl (to remove excess periodate as insoluble KIO 4 ) 13 followed by addition of O-2-NAc-D-Galp hydroxylamine (3) afforded Tn-PS A1 oxime conjugate 4 after 18 h. The 500 MHz 1 H NMR spectrum of 4 in D 2 O showed distinct oxime doublets of the Z and E isomers at 6.3 and 7.21 ppm, respectively. With 1 and 4 in hand, we proceeded to test our hypothesis in 22 C57BL/6 mice. Mice were allowed to acclimate for a 1 week period, and prior to intraperitoneal (i.p.) immunizations, mouse sera were collected (day -1). The mice were divided into four subgroups consisting of eight, six, four, and four mice (groups A, B, C, and D respectively). 1 was administered i.p. to group A, 1 plus TiterMax Gold adjuvant to group B, 4 to group C, and 4 plus TiterMax Gold adjuvant to group D. The compounds were dissolved in PBS buffer (pH 7.4) at a concentration of 10 μg/0.1 mL. On days 0, 7, 14, 21, and 28, mice were immunized with 100 μL of the appropriate solution. On days 27 and 39, blood sera from groups A-D were collected and stored at -80 °C. All of the blood sera samples were then analyzed by enzyme-linked immunosorbent assay (ELISA). PS A1-poly(L-lysine) and Tn-PS A1-poly(L-lysine) conjugates were synthesized using a well-known protocol 14 and used to coat Scheme 1. Synthesis of Tn-PS A1 Conjugate 4 10.1021/ja902607a CCC: $40.75  XXXX American Chemical Society J. AM. CHEM. SOC. XXXX, xxx, 000 9 A