Simplified Capsular Polysaccharide Isolation and Alternative Conjugation Enable the Development of a Protective Glycoconjugate Vaccine Against KL49 Acinetobacter baumannii

Background/Objectives: Acinetobacter baumannii in the intensive care setting has become increasingly carbapenem resistant in recent years. In addition to developing new antibiotics, alternative intervention strategies, such as monoclonal antibodies, bacteriophages, and vaccines, have also been explored. Capsular polysaccharides (CPS), virulence factors prominently displayed on the bacterial surface, are excellent vaccine candidates, as demonstrated by their use in preventing infections caused by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. The objective of this study is to develop and evaluate a glycoconjugate vaccine against the A. baumannii LAC-4, a K locus 49 (KL49) human clinical isolate that is hypervirulent in mice. Methods: The LAC-4 CPS was isolated and purified using a simplified base treatment method that immediately releases CPS from the anchor lipids, which also degrades DNA/RNA. Crude CPS was obtained by removal of small molecules through dialysis and proteins through phenol–water extraction. Pure K49 was obtained by SEC-HPLC on a Superose-6 column. To facilitate the separation of glycoconjugate from free polysaccharide by SEC, the CPS was depolymerized by sonication to an estimated molecular weight of 80 kDa. Because K49 lacks diol groups required for periodate oxidation, an alternative conjugation method relying on thioether bond formation rather than the commonly used reductive amination for linking K49 CPS to CRM197 was applied. Results: Immunization of mice with the conjugate vaccine elicits capsule-type-specific but strain-transcending antibody (IgM and IgG) responses. It protects immunized mice against lethal respiratory and intraperitoneal infections with both LAC-4 and other KL49 A. baumannii clinical isolates (such as BEI-24). Conclusions: To the best of our knowledge, this represents the first evaluation of a KL49 CPS glycoconjugate A. baumannii vaccine. Our results support further development of capsule-based vaccines targeting clinically important A. baumannii lineages.