Chakkumkal Anish

Chemical Biology Approaches to Designing Defined Carbohydrate Vaccines

Carbohydrate antigens have shown promise as important targets for developing effective vaccines and pathogen detection strategies. Modifying purified microbial glycans through synthetic routes or completely synthesizing antigenic motifs are attractive options to advance carbohydrate vaccine development. However, limited knowledge on structure-property correlates hampers the discovery of immunoprotective carbohydrate epitopes. Recent advancements in tools for glycan modification, high-throughput screening of biological samples, and 3D structural analysis may facilitate antigen discovery process. This review focuses on advances that accelerate carbohydrate-based vaccine development and various technologies that are driving these efforts. Herein we provide a critical overview of approaches and resources available for rational design of better carbohydrate antigens. Structurally defined and fully synthetic oligosaccharides, designed based on molecular understanding of antigen-antibody interactions, offer a promising alternative for developing future carbohydrate vaccines.

Immunological evaluation of a synthetic Clostridium difficile oligosaccharide conjugate vaccine candidate and identification of a minimal epitope.

Clostridium difficile is the cause of emerging nosocomial infections that result in abundant morbidity and mortality worldwide. Thus, the development of a vaccine to kill the bacteria to prevent this disease is highly desirable. Several recently identified bacterial surface glycans, such as PS-I and PS-II, are promising vaccine candidates to preclude C. difficile infection. To circumvent difficulties with the generation of natural PS-I due to its low expression levels in bacterial cultures, improved chemical synthesis protocols for the pentasaccharide repeating unit of PS-I and oligosaccharide substructures were utilized to produce large quantities of well-defined PS-I related glycans. The analysis of stool and serum samples obtained from C. difficile patients using glycan microarrays of synthetic oligosaccharide epitopes revealed humoral immune responses to the PS-I related glycan epitopes. Two different vaccine candidates were evaluated in the mouse model. A synthetic PS-I repeating unit CRM197 conjugate was immunogenic in mice and induced immunoglobulin class switching as well as affinity maturation. Microarray screening employing PS-I repeating unit substructures revealed the disaccharide Rha-(1→3)-Glc as a minimal epitope. A CRM197-Rha-(1→3)-Glc disaccharide conjugate was able to elicit antibodies recognizing the C. difficile PS-I pentasaccharide. We herein demonstrate that glycan microarrays exposing defined oligosaccharide epitopes help to determine the minimal immunogenic epitopes of complex oligosaccharide antigens. The synthetic PS-I pentasaccharide repeating unit as well as the Rha-(1→3)-Glc disaccharide are promising novel vaccine candidates against C. difficile that are currently in preclinical evaluation.

Glycan arrays as tools for infectious disease research

Infectious diseases cause millions of deaths worldwide each year and are a major burden for economies, especially in underdeveloped countries. Glycans and their interactions with other biomolecules are involved in all major steps of infection. Glycan arrays enable the rapid and sensitive detection of those interactions and are among the most powerful techniques to study the molecular biology of infectious diseases. This review will focus on recent developments and discuss the applications of glycan arrays to the elucidation of host-pathogen and pathogen-pathogen interactions, the development of tools for infection diagnosis and the use of glycan arrays in modern vaccine design.

Glycan arrays containing synthetic Clostridium difficile lipoteichoic acid oligomers as tools toward a carbohydrate vaccine.

Clostridium difficile is a leading cause of severe nosocomial infections. Cell-surface carbohydrate antigens are promising vaccine candidates. Here we report the first total synthesis of oligomers of the lipoteichoic acid antigen repeating unit. Synthetic glycan microarrays revealed anti-glycan antibodies in the blood of patients that help to define epitopes for vaccine development.

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.

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.

Deciphering Antigenic Determinants of Streptococcus pneumoniae Serotype 4 Capsular Polysaccharide using Synthetic Oligosaccharides

Streptococcus pneumoniae is a major cause of mortality and morbidity worldwide. More than 90 S. pneumoniae serotypes are distinguished based on the structure of their primary targets to the human immune system, the capsular polysaccharides (CPSs). The CPS of the prevalent serotype 4 (ST4) is composed of tetrasaccharide repeating units and is included in existing pneumococcal vaccines. Still, the structural antigenic determinants that are essential for protective immunity, including the role of the rare and labile cyclic trans-(2,3) pyruvate ketal modification, remain largely unknown. Molecular insights will support the design of synthetic subunit oligosaccharide vaccines. Here, we identified the key antigenic determinants of ST4 CPS with the help of pyruvated and nonpyruvated synthetic repeating unit glycans. Glycan arrays revealed oligosaccharide antigens recognized by antibodies in the human reference serum. Selected depyruvated ST4 oligosaccharides were used to formulate neoglycoconjugates and immunologically evaluated in mice. These oligosaccharides were highly immunogenic, but the resulting antiglycan antibodies showed only limited binding to the natural CPS present on the bacterial surface. Glycan array and surface plasmon resonance analysis of murine polyclonal serum antibodies as well as monoclonal antibodies revealed that terminal sugars are important in directing the immune responses. The pyruvate modification on the oligosaccharide is needed for cross-reactivity with the native CPS. These findings are an important step toward the design of oligosaccharide-based vaccines against S. pneumoniae ST4.

Antigenic Potential of a Highly Conserved Neisseria meningitidis Lipopolysaccharide Inner Core Structure Defined by Chemical Synthesis

Neisseria meningitidis is a leading cause of bacterial meningitis worldwide. We studied the potential of synthetic lipopolysaccharide (LPS) inner core structures as broadly protective antigens against N. meningitidis. Based on the specific reactivity of human serum antibodies to synthetic LPS cores, we selected a highly conserved LPS core tetrasaccharide as a promising antigen. This LPS inner core tetrasaccharide induced a robust IgG response in mice when formulated as an immunogenic glycoconjugate. Binding of raised mouse serum to a broad collection of N. meningitidis strains demonstrated the accessibility of the LPS core on viable bacteria. The distal trisaccharide was identified as the crucial epitope, whereas the proximal Kdo moiety was immunodominant and induced mainly nonprotective antibodies that are responsible for lack of functional protection in polyclonal serum. Our results identified key antigenic determinants of LPS core glycan and, hence, may aid the design of a broadly protective immunization against N. meningitidis.

Chemical Synthesis Elucidates the Immunological Importance of a Pyruvate Modification in the Capsular Polysaccharide of Streptococcus pneumoniae Serotype 4

Carbohydrate modifications are believed to strongly affect the immunogenicity of glycans. Capsular polysaccharides (CPS) from bacterial pathogens are frequently equipped with a pyruvate that can be placed across the 4,6-, 3,4-, or 2,3-positions. A trans-2,3-linked pyruvate is present on the CPS of the Gram-positive bacterium Streptococcus pneumoniae serotype 4 (ST4), a pathogen responsible for pneumococcal infections. To assess the immunological importance of this modification within the CPS repeating unit, the first total synthesis of the glycan was carried out. Glycan microarrays containing a series of synthetic antigens demonstrated how antibodies raised against natural ST4 CPS specifically recognize the pyruvate within the context of the tetrasaccharide repeating unit. The pyruvate modification is a key motif for designing minimal synthetic carbohydrate vaccines for ST4.

The immunogenic characteristics associated with multivalent display of Vi polysaccharide antigen using biodegradable polymer particles.

Polysaccharides in their great majority are thymus-independent (TI) antigens. Anti-polysaccharide antibody responses are generally weak and characterized by lack of memory, isotype restriction and delayed ontogeny. We report here the generation of protective memory antibody response by multivalent display of polysaccharide antigens on biodegradable polymeric particles. Single dose immunization using polylactide (PLA) polymer particles entrapping Vi capsular polysaccharide antigen from Salmonella typhi promoted isotype switching and induced polysaccharide-specific memory antibody response in experimental animals. PLA nanoparticles as well as microparticles entrapping Vi polysaccharides elicited high IgG titer in comparison to soluble Vi immunization. Immunizations with particles co-entrapping both Vi polysaccharide and tetanus toxoid did not improve the anti-polysaccharide antibody responses. Lower antibody response from co-entrapped formulation was mostly due to inhibition of particle phagocytosis by the macrophages. Immunization using polylactide particles entrapping only Vi polysaccharide with higher density on surface elicited highest secondary antibody response as well as promoted isotype switching. The vaccination potential of particle based immunizations was further confirmed by the generation of quick memory antibody responses while challenging the immunized animals with live S. typhi. This approach provides a multivalent display of polysaccharide antigen using polymer particles and elicits protective memory antibody response without conjugation to a carrier protein.