Development of a new trend conjugate vaccine for the prevention of Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae is a major cause of nosocomial pneumonia, septicemia and urinary tract infections, especially in newborns, blood cancer patients, and other immunocompromised candidates. The control of K. pneumoniae is a complicated issue due to its tight pathogenesis. Immuno-prophylactic preparations, especially those directed toward the bacterium O-antigen, showed to be the most successful way to prevent the infection incidence. However, all previously proposed preparations were either of limited spectrum or non-maternal, and hence not targeting the main Klebsiella patients. Moreover, all preparations were directed only to prevent the respiratory diseases due to that pathogen. This article addresses the development of a method originally used to purify the non-capsular bacterial-endotoxins, as a new and easy method for vaccine production against K. pneumoniae. The application of this method was preceded by a biotechnological control of capsular polysaccharide production in K. pneumoniae. The new produced natural conjugate between the bacterial O-antigen and its outer membrane proteins was evaluated by physicochemical and immunological methods to investigate its purity, integrity, safety and immunogenicity. It showed to be pure, stable, safe for use, and able to elicit a protective immunoglobulin titer against different Klebsiella infections. This immune-response proved to be transferable to the offspring of the vaccinated experimental rabbits via placenta.

Keywords: K. pneumonia; conjugate vaccines; isobutyric acid; lipo-polysaccharides..

Figure 1

Twelve percent sulfate-polyacrylamide gel electrophoresis stained by Alcian blue for the biomass; in the presence of 10, 20, 30, 40 and 50 µg/mL medium, sodium salicylate (from left to right). The blue smear indicates the presence of capsular polysaccharide. The gel was run at 120 V for 150 minutes.

Figure 2

Refractive index chart of the glycopeptide, on high performance liquid chromatography using pure acetonitrile at flow rate of 1 mL min⁻¹.

Figure 3

Ultra-violet 280 nm chart of the glycopeptide, on high performance liquid chromatography using pure acetonitrile at flow rate of 1 mL min⁻¹. The two main peaks description is provided and shows their relative proportion.

Figure 4

Enzyme-linked immuno-sorbent assay antibody titer monitored all over 7 weeks for rabbits groups: GP.1 control group, GP.2 study group with different concentrations of glycopeptide injection (50, 100, and 150 µg/kg). The absorbance of the developing color due to horse-radish peroxidase labeled polyvalent goat-anti-rabbit immunoglobulin substrate (Sigma-Aldrish, USA) was read at 450 nm, data were analyzed by three-ways analysis of variance statistics that revealed a P-value of less than 0.005 for the read absorbance replica.