Tolerance and Persistence of Ebola Virus in Primary Cells from Mops condylurus, a Potential Ebola Virus Reservoir.

Although there have been documented Ebola virus disease outbreaks for more than 40 years, the natural reservoir host has not been identified. Recent studies provide evidence that the Angolan free-tailed bat (Mops condylurus), an insectivorous microbat, is a possible ebolavirus reservoir. To investigate the potential role of this bat species in the ecology of ebolaviruses, replication, tolerance, and persistence of Ebola virus (EBOV) were investigated in 10 different primary bat cell isolates from M. condylurus. Varying EBOV replication kinetics corresponded to the expression levels of the integral membrane protein NPC1. All primary cells were highly tolerant to EBOV infection without cytopathic effects. The observed persistent EBOV infection for 150 days in lung primary cells, without resultant selective pressure leading to virus mutation, indicate the intrinsic ability of EBOV to persist in this bat species. These results provide further evidence for this bat species to be a likely reservoir of ebolaviruses.

Development of an Efficacious, Semisynthetic Glycoconjugate Vaccine Candidate against Streptococcus pneumoniae Serotype 1.

Infections with Streptococcus pneumoniae are a major health burden. Glycoconjugate vaccines based on capsular polysaccharides (CPSs) successfully protect from infection, but not all pneumococcal serotypes are covered with equal potency. Marketed glycoconjugate vaccines induce low levels of functional antibodies against the highly invasive serotype 1 (ST1), presumably due to the obscuring of protective epitopes during chemical activation and conjugation to carrier proteins. Synthetic oligosaccharide antigens can be designed to carry linkers for site-selective protein conjugation while keeping protective epitopes intact. Here, we developed an efficacious semisynthetic ST1 glycoconjugate vaccine candidate. A panel of synthetic oligosaccharides served to reveal a critical role of the rare aminosugar, 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose (d-AAT), for ST1 immune recognition. A monovalent ST1 trisaccharide carrying d-AAT at the nonreducing end induced a strong antibacterial immune response in rabbits and outperformed the ST1 component of the multivalent blockbuster vaccine Prevenar 13, paving the way for a more efficacious vaccine.

A semisynthetic Streptococcus pneumoniae serotype 8 glycoconjugate vaccine.

Glycoconjugate vaccines based on capsular polysaccharides (CPSs) of pathogenic bacteria such as Streptococcus pneumoniae successfully protect from disease but suffer from incomplete coverage, are troublesome to manufacture from isolated CPSs, and lack efficacy against certain serotypes. Defined, synthetic oligosaccharides are an attractive alternative to isolated CPSs but require the identification of immunogenic and protective oligosaccharide antigens. We describe a medicinal chemistry strategy based on a combination of automated glycan assembly (AGA), glycan microarray-based monoclonal antibody (mAb) reverse engineering, and immunological evaluation in vivo to uncover a protective glycan epitope (glycotope) for S. pneumoniae serotype 8 (ST8). All four tetrasaccharide frameshifts of ST8 CPS were prepared by AGA and used in glycan microarray experiments to identify the glycotopes recognized by antibodies against ST8. One tetrasaccharide frameshift that was preferentially recognized by a protective, CPS-directed mAb was conjugated to the carrier protein CRM197. Immunization of mice with this semisynthetic glycoconjugate followed by generation and characterization of a protective mAb identified protective and nonprotective glycotopes. Immunization of rabbits with semisynthetic ST8 glycoconjugates containing protective glycotopes induced an antibacterial immune response. Coformulation of ST8 glycoconjugates with the marketed 13-valent glycoconjugate vaccine Prevnar 13 yielded a potent 14-valent S. pneumoniae vaccine. Our strategy presents a facile approach to develop efficient semisynthetic glycoconjugate vaccines.

A Semi-synthetic Oligosaccharide Conjugate Vaccine Candidate Confers Protection against Streptococcus pneumoniae Serotype 3 Infection.

The identification of immunogenic glycotopes that render glycoconjugate vaccines protective is key to improving vaccine efficacy. Synthetic oligosaccharides are an attractive alternative to the heterogeneous preparations of purified polysaccharides that most marketed glycoconjugate vaccines are based on. To investigate the potency of semi-synthetic glycoconjugates, we chose the least-efficient serotype in the current pneumococcal conjugate vaccine Prevnar 13, Streptococcus pneumoniae serotype 3 (ST3). Glycan arrays containing synthetic ST3 repeating unit oligosaccharides were used to screen a human reference serum for antibodies and to define the recognition site of two ST3-specific protective monoclonal antibodies. The glycan array screens identified a tetrasaccharide that was selected for in-depth immunological evaluation. The tetrasaccharide-CRM197 carrier protein conjugate elicited protective immunity as evidenced by opsonophagocytosis assays and protection against pneumonia caused by ST3 in mice. Formulation of the defined protective lead candidate glycotope has to be further evaluated to elicit optimal long-term immunity.

Multivalent display of minimal Clostridium difficile glycan epitopes mimics antigenic properties of larger glycans.

Synthetic cell-surface glycans are promising vaccine candidates against Clostridium difficile. The complexity of large, highly antigenic and immunogenic glycans is a synthetic challenge. Less complex antigens providing similar immune responses are desirable for vaccine development. Based on molecular-level glycan-antibody interaction analyses, we here demonstrate that the C. difficile surface polysaccharide-I (PS-I) can be resembled by multivalent display of minimal disaccharide epitopes on a synthetic scaffold that does not participate in binding. We show that antibody avidity as a measure of antigenicity increases by about five orders of magnitude when disaccharides are compared with constructs containing five disaccharides. The synthetic, pentavalent vaccine candidate containing a peptide T-cell epitope elicits weak but highly specific antibody responses to larger PS-I glycans in mice. This study highlights the potential of multivalently displaying small oligosaccharides to achieve antigenicity characteristic of larger glycans. The approach may result in more cost-efficient carbohydrate vaccines with reduced synthetic effort.

Epitope recognition of antibodies against a Yersinia pestis lipopolysaccharide trisaccharide component.

Today, the process of selecting carbohydrate antigens as a basis for active vaccination and the generation of antibodies for therapeutic and diagnostic purposes is based on intuition combined with trial and error experiments. In efforts to establish a rational process for glycan epitope selection, we employed glycan array screening, surface plasmon resonance, and saturation transfer difference (STD)-NMR to elucidate the interactions between antibodies and glycans representing the Yersinia pestis lipopolysaccharide (LPS). A trisaccharide epitope of the LPS inner core glycan and different LPS-derived oligosaccharides from various Gram-negative bacteria were analyzed using this combination of techniques. The antibody-glycan interaction with a heptose substructure was determined at atomic-level detail. Antibodies specifically recognize the Y. pestis trisaccharide and some substructures with high affinity and specificity. No significant binding to LPS glycans from other bacteria was observed, which suggests that the epitopes for just one particular bacterial species can be identified. On the basis of these results we are beginning to understand the rules for structure-based design and selection of carbohydrate antigens.

Immunogenicity and diagnostic potential of synthetic antigenic cell surface glycans of Leishmania.

Detection and quantification of pathogen-derived antigenic structures is a key method for the initial diagnosis and follow-up of various infectious diseases. Complex parasitic diseases such as leishmaniasis require highly sensitive and specific tests prior to treatment with potentially toxic drugs. To investigate the diagnostic potential of cell surface glycans found on Leishmania parasites, we identified diagnostically relevant glycan epitopes and used synthetic glycan microarrays to screen sera from infected humans and dogs. On the basis of the screening results, we selected a tetrasaccharide to generate anti-glycan antibodies. The corresponding tetrasaccharide-carrier protein conjugate was immunogenic in mice, and sera obtained from immunized mice specifically detected the Leishmania parasite. These results demonstrate how synthetic glycan arrays, in combination with immunological methods, help to identify promising carbohydrate antigens for pathogen detection.