A scalable method for O-antigen purification applied to various Salmonella serovars.

The surface lipopolysaccharide of gram-negative bacteria is both a virulence factor and a B cell antigen. Antibodies against O-antigen of lipopolysaccharide may confer protection against infection, and O-antigen conjugates have been designed against multiple pathogens. Here, we describe a simplified methodology for extraction and purification of the O-antigen core portion of Salmonella lipopolysaccharide, suitable for large-scale production. Lipopolysaccharide extraction and delipidation are performed by acetic acid hydrolysis of whole bacterial culture and can take place directly in a bioreactor, without previous isolation and inactivation of bacteria. Further O-antigen core purification consists of rapid filtration and precipitation steps, without using enzymes or hazardous chemicals. The process was successfully applied to various Salmonella enterica serovars (Paratyphi A, Typhimurium, and Enteritidis), obtaining good yields of high-quality material, suitable for conjugate vaccine preparations.

Production of a conjugate vaccine for Salmonella enterica serovar Typhi from Citrobacter Vi.

A conjugate vaccine for Salmonella enterica serovar Typhi was produced by chemically linking Vi, purified from Citrobacter, to the non-toxic mutant diphtheria toxin CRM(197) via an adipic dihydrazide spacer using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide coupling chemistry. The polysaccharide purification process was developed based on Vi precipitation from culture supernatant with cetyl trimethylammonium bromide (CTAB), solubilization of the CTA-polysaccharide salt with ethanol followed by exchange of the CTA(+) counter ion with Na(+). The purified Vi polysaccharide was fully O-acetylated and with high purity. The conjugation process was optimized to obtain a scalable process that has been used for GMP production at pilot scale of vaccine currently in clinical trials.

Vi-CRM 197 as a new conjugate vaccine against Salmonella Typhi.

An efficacious, low cost vaccine against typhoid fever, especially for young children, would make a major impact on disease burden in developing countries. The virulence capsular polysaccharide of Salmonella Typhi (Vi) coupled to recombinant mutant Pseudomonas aeruginosa exoprotein A (Vi-rEPA) has been shown to be highly efficacious. We investigated the use of carrier proteins included in infant vaccines, standardized the conjugation process and developed key assays required for routine lot release at production scale. Vi from a BSL1 organism, Citrobacter freundii, strain WR7011, was used as an alternative to Vi from S. Typhi. We showed that Vi conjugated to CRM(197), a non-toxic mutant of diphtheria toxin, widely used in commercial vaccines, was produced at high yield. Vi-CRM(197) proved immunogenic in animal studies, even without adjuvant. Thus, Vi-CRM(197) appears to be a suitable candidate for the development of a commercially viable, effective typhoid vaccine for developing countries.

Continuous ambulatory peritoneal dialysis and sclerosing encapsulating peritonitis: tamoxifen as a new therapeutic agent?

Sclerosing encapsulating peritonitis (SEP) is a serious complication of long-term continuous ambulatory peritoneal dialysis (CAPD), very likely related to a persisting expression of the transforming growth factor beta1 (TGFbeta1) gene on peritoneal mesothelial cells. We report the case of a 67-year-old uremic woman who developed SEP eight years after being placed on CAPD, complicated by eight episodes of bacterial peritonitis. CAPD was therefore stopped and the patient transferred to hemodialysis. The diagnosis of SEP was confirmed by physical findings (vomiting, abdominal pain with palpable mass, ileus, cachexia) and CT data. The patient was treated with tamoxifen (10 mg/day) for three months, and gradually recovered, a subsequent CT showing a significant reduction of the thickness of peritoneal and intestinal loops. Tamoxifen probably interferes with TGFbeta1 and may be useful in the treatment of this CAPD complication.

Continuous venous-venous hemofiltration for the treatment of fluid overload in continuous ambulatory peritoneal dialysis patients.

Fluid overload is not infrequent in continuous ambulatory peritoneal dialysis (CAPD) patients. In our experience, extemporaneous continuous venous-venous hemofiltration (CVVHF) was able to correct fluid imbalances refractory to high dose diuretics and hypertonic solutions. We treated 8 of 52 patients (5 females, 3 males, mean age 52 years) on CAPD from 4 to 36 months and with fluid overloads of up to 10 kg. A Biospal SCU/CAVH flat-sheet high-flux hemodialyzer employed for 10 h produced an ultrafiltration rate (QB:150 ml/min) of 11.12 +/- 4.97 ml/min. With an isotonic replacement solution, the filter provided sufficient extraction of small molecules so that CAPD could be interrupted during CVVHF. The procedure appeared well tolerated. This approach reduced the use of hypertonic dialysate, which is not devoid of side effects on ultrafiltration capacity of the peritoneal membrane.