Effect of saccharide length on the immunogenicity of neoglycoconjugates from synthetic fragments of the O-SP of Vibrio cholerae O1, serotype Ogawa Rina Saksena, a Xingquan Ma, a Terri K. Wade, b Pavol Kova ´c ˇ a and William F. Wade b, * a National Institutes of Health, NIDDK, Laboratory of Medicinal Chemistry, Bethesda, MD 20892, USA b Department of Microbiology and Immunology, Dartmouth Medical School, 630W. Borwell Bldg., Lebanon, NH 03756, USA Received 6 June 2005; received in revised form 22 July 2005; accepted 24 July 2005 Available online 10 August 2005 Abstract—A synthetic hexasaccharide, identical to the terminal hexasaccharide of Ogawa LPS, coupled to bovine serum albumin induced protective antibodies in mice. To determine if there was a minimum saccharide length required for immunogenicity and efficacy, shorter (mono- to pentasaccharide) neoglycoconjugates (CHO–BSA) were tested in mice. The Ogawa CHO–BSA was inocu- lated at either a constant mass but differing moles, or equal moles but differing masses. Humoral responses were essentially the same when mice received 9 lg of the carbohydrate (0.007 mM with the pentasaccharide) in each of the neoglycoconjugates prepared from mono- through the pentasaccharide, or the same molar amount (0.007 mM), proportionally less by weight when going from the penta- to the monosaccharide. These data show that, within this dose range, the responses occurred virtually independently of the amount of immunogen. Humoral antibodies induced by these immunogens were generally not vibriocidal. Selected antisera induced by CHO–BSA immunogens were protective, but the ELISA titers of the sera were not predictive of the protective capacity. Purified, Ogawa LPS induced anti-Ogawa LPS IgM antibody titers similar to those induced by the Ogawa CHO–BSA conjugates. The anti-whole LPS sera were strongly vibriocidal, as were the previously reported sera induced by hexasaccharide conjugates. This suggests either that the shorter oligosaccharides lack a conformational epitope provided by the hexasaccharide or that the LPS has additional B cell epitopes or selects different B cells in the primary response. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Vibrio cholerae O1; Cholera vaccine; Ogawa; LPS; Conjugate; Neoglycoconjugate 1. Introduction Cholera is a diarrheal disease caused by the gram-nega- tive bacterium Vibrio cholerae. LPS from V. cholerae O1 serogroup (a serogroup is a collection of serotypes with different structures within the LPS defined by antibo- dies) induces protective immune responses in humans and experimental animals. 1–3 V. cholerae-specific, anti- LPS antibodies are correlated with protection against cholera 2 and, thus, LPS is an immunogen of choice for cholera vaccine development. An immunogen is a mac- romolecule capable of inducing an immune response. The immunogenicity of a particular immunogen, does not necessarily correlate with its ability to induce protec- tive antibodies and thus immunogenicity must be associ- ated with efficacy. Multiple monoclonal antibodies 4–8 developed against V. cholerae LPS to define the serotypes (structural iden- tity of the LPS) of clinical isolates suggests that there are multiple B cell epitopes (antibody binding targets, protective or not) expressed by the two most prevalent serotypes of V. cholerae LPS, Ogawa and Inaba. Three 0008-6215/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2005.07.017 Abbreviations: ELISA, enzyme-linked immunosorbant assay; ANOVA, analysis of variance; IgM, immunoglobulin M; IgG1, immunoglobulin G1; LPS, lipopolysaccharide; O-SP, O-specific polysaccharide; CHO– BSA, carbohydrate–bovine serum albumin conjugate. * Corresponding author. Tel.: +1 603 650 6896; fax: +1 603 650 6223; e-mail: william.wade@dartmouth.edu Carbohydrate RESEARCH Carbohydrate Research 340 (2005) 2256–2269