Synthesis and Evaluation of a Bivalent Glycoconjugate Vaccine for Prevention of Infection with Acinetobacter baumannii

The proposed work will involve the synthesis and evaluation of glycoconjugates designed to trigger adaptive immune responses that provide protection against infection by the bacteria Acinetobacter baumannii. Drug resistance to this pathogen is on the rise, and there are no approved vaccines to prevent infection.

Central Hypothesis: Conjugation of two classes of glycan from the surface of A. baumannii to so-called carrier proteins will result in an effective glycoconjugate vaccine with broad coverage against various strains. Project Goals: Short term goals include the synthesis of two classes of glycoconjugate inspired by A.

baumannii glycans and their injection into mice to determine their immunogenicity and protective effects against infection. Long-term goals include the development of a binary glycoconjugate vaccine (based on the two classes of glycan) that can prevent infection with A. baumannii in humans. Specific Aims: Aim 1:

Chemical Synthesis of A. baumannii glycans. Both a pentasaccharide O-glycan and fragments of a lipooligosaccharide (LOS) from A. baumannii will be synthesized chemically. Successful execution of this Aim will result in the production of homogeneous and conjugation-ready glycans. Aim 2: Covalent conjugation of

A. baumannii Glycans to carrier Proteins. Glycans from Aim 1 will be equipped with a linker and then covalently linked to, inter alia, the carrier protein CRM197. Successful execution of this Aim will result in well- characterized glycoconjugates for immunological study. Aim 3: Immunological Evaluation of the

Glycoconjugates. Mice will be immunized with the glycoconjugates. The antigenicity of these glycoconjugates will be studied. Immunized mice will then be challenged with A. baumannii to determine the protective effects of immunization and to study the nature of the immune response. Successful execution of this work will result

in high titers of antibodies and robust protection of immunized mice relative to controls. Specific Aim 1 studies will involve multistep chemical synthesis of a series of 3 LOS fragments of varying complexity. We have completed the synthesis of the aforementioned O-glycan and one LOS fragment already. Key aspects of these syntheses include “endgame” assembly of oligosaccharides and protecting

group removal. Specific Aim 2 studies will involve appendage of the O-glycan and LOS fragments with e.g. thiol or alkoxyamine linkers and subsequent conjugation to pre-functionalized derivatives of the carrier protein CRM197 through sulfide or oxime linkages, respectively. Specific Aim 3 studies will involve immunization of

mice with the conjugates. Anti-glycan antibody titers in the resulting immune sera will be determined with ELISA, and binding of the antibodies to A. baumannii will be studied using flow cytometry. Immunized mice will be challenged with A. baumannii, and cytokine profiling will be conducted to study the nature of the immune

response while the extent of protection will be determined by studying survival rates and bacterial burden. Such challenge experiments will be conducted with mice immunized with the most promising glycoconjugates.