The capsular saccharides of bacteria have been used for many years in vaccines against capsulated bacteria. As saccharides are T-independent antigens, however, they are poorly immunogenic. Conjugation to a carrier can convert T-independent antigens into T-dependent antigens, thereby enhancing memory responses and allowing protective immunity to develop. The most effective saccharide vaccines are therefore based on glycoconjugates, and the prototype conjugate vaccine was against Haemophilus influenzae type b (‘Hib’) [e.g. see chapter 14 of ref. 96].
Another bacterium for which conjugate vaccines have been described is Staphylococcus aureus (S. aureus). Various polysaccharides have been isolated from S. aureus for use in glycoconjugates. Two polysaccharides of particular interest are the type 5 and type 8 capsular polysaccharides. Approximately 60% of human S. aureus strains are type 8 and approximately 30% are type 5. Much of the work on type 5 and type 8 conjugates has been performed by Fattom et al., and is described in documents such as references 1 to 9.
The starting point for polysaccharide-based vaccines is the polysaccharide itself, and this is generally purified from the target bacterium. Fattom et al. have developed a complex process for purification of the type 5 and type 8 capsular polysaccharides that is described in detail in reference 1, and involves the following key steps after bacterial culture: suspension of bacterial cells in buffer, treatment with lysostaphin, treatment with DNase and RNase, centrifugation, dialysis against buffer, treatment with protease, further dialysis, filtration, addition of ethanol to 25% with calcium chloride to precipitate contaminants; further addition of ethanol to 75% to precipitate the polysaccharide; collection and drying of the precipitate; anion exchange chromatography; dialysis; lyophilisation; size exclusion chromatography; dialysis and final lyophilisation.
The Fattom process involves the use of lysostaphin to lyse the bacterial cell walls and thereby release capsular polysaccharide. However, this step is time-consuming and makes the process difficult to scale-up to an industrial setting. It also increases the overall cost and complexity of the process. Other researchers have attempted to omit this step and develop a simpler, more efficient method of purifying the polysaccharide. For example, reference [10] describes an alternative process that involves autoclaving S. aureus cells, ultrafiltration of the polysaccharide-containing supernatant, concentration, lyophilisation, treatment with sodium metaperiodate, further ultrafiltration, diafiltration, high performance size exclusion liquid chromatography, dialysis and freeze-drying. The authors suggest that this method provides a good yield and is suitable for large scale production of polysaccharide. In this method, the lysostaphin treatment is replaced by autoclaving to release capsular polysaccharide. The method was further developed in reference [11]. An important step in these alternative methods is the treatment with sodium metaperiodate. This step is carried out to remove teichoic acid contamination of the capsular polysaccharide. However, once again this step increases the duration, complexity and overall cost of the process. Reference [12] describes a similar process that again involves autoclaving to release capsular polysaccharide and treatment with sodium metaperiodate to remove teichoic acid. In contrast, most other groups use processes that retain lysostaphin treatment (see, for example, references 13, 14, 15, 16, 17 and 18), sometimes including treatment with sodium metaperiodate (e.g. in references 13 and 14).
The above methods are complex and may leave contamination in the resultant polysaccharide. There is thus a need for further and improved processes for purifying S. aureus type 5 and type 8 capsular polysaccharides, and particularly for less complex processes that result in less contamination.
USPTO Backgrounds
USPTO Backgrounds