The realm of carbohydrate vaccines has expanded far beyond the capsular polysaccharides of bacterial pathogens to include a diverse collection of targets representing nearly every biological kingdom. Recent technological advances in glycobiology and glycochemistry are paving the way for a new era in carbohydrate vaccine design enabling greater efficiency in the identification, synthesis and evaluation of unique glycan epitopes found on a plethora of pathogens and malignant cells. This article reviews the progress being made in addressing challenges posed by targeting the surface carbohydrates of bacteria, protozoa, helminths, viruses, fungi and cancer for vaccine purposes.

Human antibody 2G12 neutralizes a broad range of HIV-1 isolates. Hence, molecular characterization of its epitope, which corresponds to a conserved cluster of oligomannoses on the viral envelope glycoprotein gp120, is a high priority in HIV vaccine design. A prior crystal structure of 2G12 in complex with Man 9GlcNAc2 highlighted the central importance of the D1 arm in antibody binding. To characterize the specificity of 2G12 more precisely, we performed solution-phase ELISA, carbohydrate microarray analysis, and cocrystallized Fab 2G12 with four different oligomannose derivatives (Man 4, Man5, Man7, and Man8) that compete with gp120 for binding to 2G12. Our combined studies reveal that 2G12 is capable of binding both the D1 and D3 arms of the Man 9GlcNAc2 moiety, which would provide more flexibility to make the required multivalent interactions between the antibody and the gp120 oligomannose cluster than thought previously. These results have important consequences for the design of immunogens to elicit 2G12-like neutralizing antibodies as a component of an HIV vaccine. 2G12 antibody ͉ HIV vaccine ͉ oligomannoses There is widespread agreement that the most promising approach to contain the ongoing HIV pandemic is through the development of an effective vaccine (1, 2). However, HIV vaccine design has faced many difficulties including, prominently, the lack of an immunogen able to elicit broadly neutralizing antibodies (Abs). The feasibility of developing such an immunogen is suggested by a small panel of broadly neutralizing human monoclonal antibodies (mAbs) that have been isolated from seropositive donors (3). One of these Abs, 2G12, recognizes a conserved and unusually dense cluster of oligomannose residues on the ''silent face'' of gp120, the major envelope protein of HIV-1 (4, 5). Many lectins have been identified that can bind to envelope and act antivirally, but 2G12 remains the only known anti-carbohydrate protein that has been specifically elicited to HIV-1 in an adaptive immune response (6). 2G12 has an unusual architecture in which the arms of the IgG swap variable heavy domains, creating a domainswapped dimer of Fabs (7). The crystal structure of Fab 2G12 complexed with Man 9 GlcNAc 2 indicated that the conventional Ab-binding sites are occupied by the D1 arms of the Man 9 GlcNAc 2 moieties (1; Fig. 1) (7). The terminal Man␣1-2Man residues of the D1 branch account for 85% of the Fab contacts to Man 9 GlcNAc 2 , although the disaccharide by itself is 50-fold less potent in binding to 2G12 than Man 9 GlcNAc 2 (7).Based on these structural results, several initiatives have been launched to design novel immunogens that will elicit 2G12-like Abs (8-12). Recently, we described the design and synthesis of novel antigens, oligomannoses 2-6 (Fig. 1), that bind to mAb 2G12 (13). The Man␣1-2Man-containing oligomannoses 4, 5, and 6 were identified as new epitope mimics that inhibit the binding of gp120 to mAb 2G12 as well as, or better than, Man 9 GlcNAc 2 (13). Encouraged by this result, we now report the desig...

It is widely accepted that the heavily glycosylated glycoprotein gp120 on the surface of HIV-1 shields peptide epitopes from recognition by the immune system and may promote infection in vivo by interaction with dendritic cells and transport to tissue rich in CD4 ؉ T cells such as lymph nodes. A conserved cluster of oligomannose glycans on gp120 has been identified as the epitope recognized by the broadly HIV-1-neutralizing monoclonal antibody 2G12. Oligomannose glycans are also the ligands for DC-SIGN, a C-type lectin found on the surface of dendritic cells. Multivalency is fundamental for carbohydrate-protein interactions, and mimicking of the high glycan density on the virus surface has become essential for designing carbohydrate-based HIV vaccines and antiviral agents. We report an efficient synthesis of oligomannose dendrons, which display multivalent oligomannoses in high density, and characterize their interaction with 2G12 and DC-SIGN by a glycan microarray binding assay. The solution and the surface binding analysis of 2G12 to a prototype oligomannose dendron clearly demonstrated the efficacy of dendrimeric display. We further showed that these glycodendrons inhibit the binding of gp120 to 2G12 and recombinant dimeric DC-SIGN with IC 50 in the nanomolar range. A second-generation Man 9 dendron was identified as a potential immunogen for HIV vaccine development and as a potential antiviral agent.glycodendron ͉ high mannose ͉ multivalency ͉ HIV vaccine ͉ antiviral agent

Summary The broadly neutralizing antibody 2G12 recognizes a conserved cluster of high mannose glycans on the surface envelope spike of HIV suggesting that the “glycan shield” defense of the virus can be breached and may, under the right circumstances, serve as a vaccine target. In an attempt to recreate features of the glycan shield semi-synthetically, oligomannosides were coupled to surface lysines on the icosahedral capsids of bacteriophage Qβ and cowpea mosaic virus (CPMV). The Qβ glycoconjugates, but not CPMV, presented oligomannose clusters that bind the antibody 2G12 with high affinity. However, Abs against these 2G12 epitopes were not detected in immunized rabbits. Rather, alternative oligomannose epitopes on the conjugates were immunodominant and elicited high titres of anti-mannose Abs that do not cross-react with the HIV envelope. The results presented reveal important design considerations for a carbohydrate-based vaccine component for HIV.

The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly neutralizing antibody (Ab), 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G12 raises the possibility that a carbohydrate immunogen may be developed that could elicit 2G12-like neutralizing Abs and contribute to an AIDS vaccine. We have previously dissected the fine specificity of 2G12 and reported that the synthetic tetramannoside (Man 4 ) that corresponds to the D1 arm of Man 9 GlcNAc 2 inhibits 2G12 binding to gp120 as efficiently as Man 9 GlcNAc 2 itself, indicating the potential use of Man 4 as a building block for creating immunogens. Here, we describe the development of neoglycoconjugates displaying variable copy numbers of Man 4 on bovine serum albumin (BSA) molecules by conjugation to Lys residues. The increased valency enhances the apparent affinity of 2G12 for Man 4 up to a limit which is achieved at ϳ10 copies per BSA molecule, beyond which no further enhancement is observed. Immunization of rabbits with BSA-(Man 4 ) 14 elicits significant serum Ab titers to Man 4 . However, these Abs are unable to bind gp120. Further analysis reveals that the elicited Abs bind a variety of unbranched and, to a lesser extent, branched Man 9 derivatives but not natural N-linked oligomannose containing the chitobiose core. These results suggest that Abs can be readily elicited against the D1 arm; however, potential differences in the presentation of Man 4 on neoglycoconjugates, compared to glycoproteins, poses challenges for eliciting anti-mannose Abs capable of crossreacting with gp120 and HIV-1.