Synthesis and characterization of Pseudomonas aeruginosa alginate–tetanus toxoid conjugate

Abstract

Chronic infection with Pseudomonas aeruginosa is the main proven perpetrator of lung function decline and ultimate mortality in cystic fibrosis (CF) patients. Mucoid strains of this bacterium elaborate mucoid exopolysaccharide, also referred to as alginate. Alginate-based immunization of naïve animals elicits opsonic antibodies and leads to clearance of mucoid P. aeruginosa from the lungs. Alginate was isolated from mucoid P. aeruginosa strain 8821M by repeated ethanol precipitation, dialysis, proteinase and nuclease digestion, and chromatography. To improve immunogenicity, the purified antigen was coupled to tetanus toxoid (TT) with adipic acid dihydrazide (ADH) as a spacer and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as a linker. The reaction mixture was passed through a Sepharose CL-4B column. The resulting conjugate was composed of TT and large-size alginate polymer at a ratio of about 3 : 1; it was non-toxic and non-pyrogenic, and elicited high titres of alginate-specific IgG. Antisera raised against the conjugate had high opsonic activity against the vaccine strain. The alginate conjugate was also able to protect mice against a lethal dose of mucoid P. aeruginosa. These data indicate that an alginate-based vaccine has significant potential to protect against chronic infection with mucoid P. aeruginosa in the CF host.

Efficacy of a Conjugate Vaccine Containing Polymannuronic Acid and Flagellin against Experimental Pseudomonas aeruginosa Lung Infection in Mice

“…Specificity of antisera to PMA and PMA-FLA for alginate was shown by its inability to promote opsonic killing of an algD mutant strain of FRD1. Previously published data demonstrated that immunization with native P. aeruginosa alginate can induce opsonic antibodies to mucoid P. aeruginosa (15,20,35), and other groups found that conjugation of alginate to carrier proteins significantly increased the opsonic killing activity of mucoid strains (10,20,47). Likely, the conjugation of PMA to flagellin enhanced its immunogenicity, as lower doses of the conjugate were needed compared with the purified carbohydrate antigen alone in order to induce opsonic killing antibody.…”

“…Vaccination to prevent infection by Pseudomonas aeruginosa would be a welcome advance in many regards, but particularly for preventing the chronic infection that is the major cause of morbidity and mortality in cystic fibrosis (CF) patients (20,24). Colonization of the CF lung by P. aeruginosa occurs early in life (23,42) predominantly by low-alginate-producing, nonmucoid, motile strains expressing a single polar flagellum.…”

“…Purified P. aeruginosa alginate is safe when administered to humans (35) but alginate overall is poorly immunogenic in most humans, failing to elicit high titers of opsonic, protective antibodies. In an attempt to increase the immunogenicity of alginate, the molecule has been conjugated to carrier proteins (10,13,20,47) by using detoxified exotoxin A, keyhole limpet hemocyanin (KLH), and tetanus toxoid (TT) as carrier proteins. However, a major challenge to this approach is that alginate from different mucoid strains has variable ratios of mannuronic to guluronic acids (ϳ10:1 to close to 1:1) (35), making it difficult to find a preparation of alginate that gives rise to antibodies reactive with multiple strains of mucoid P. aeruginosa.…”

Shrimp pathogenicity, hemolysis, and the presence of hemolysin and TTSS genes in Vibrio harveyi isolated from Thailand

“…The LD 50 of V. harveyi was calculated using the method of Reed & Muench (1938). For evaluation of virulence of all Vibrio harveyi isolates obtained from fish and shellfish, each isolate was injected into shrimps at a concentration of 4 × LD 50 (Kashef et al 2006), using the same procedure as described in the previous paragraph. Mortality rates were measured 48 h after injection.…”

Prevention of Pseudomonas aeruginosa infection in cystic fibrosis patients

“…No data on animal protection have been published yet, however, preclinical trials with the KLH conjugate are ongoing. Finally, a P. aeruginosa alginate-tetanus toxoid conjugate vaccine has been produced which protected mice against a lethal dose of an alginate-overproducing P. aeruginosa strain (Kashef et al, 2006).…”