Active Vaccination Against Bacterial Colonization
The first stage of a microbial infection is colonization, and pathogens have developed strategies to efficiently invade or attach to host tissues. Virulence factors involved in this process, therefore, represent interesting targets for vaccine development. This approach could complement antitoxin strategies, because colonization of the gut and adhesion to mucosal surfaces precedes toxin production. Important efforts are currently being undertaken to identify factors controlling tissue invasion by C. difficile, and more than 30 cell wall proteins have been identified. While the contribution to virulence and the exposure at the surface of the pathogen for most cell wall proteins is yet to be established, some of them have already been characterized in detail and tested as vaccine candidates that might inhibit colonization and adhesion of the bacteria to gut epithelia. In C. difficile, two proteins encoded by a single gene, SlpA, cover most of the bacteria cell surface. A LMW-Slp that is variable and attached to the external surface of the bacteria and a HMW-Slp that is more conserved and linked to the LMW-Slp. Anti-Slp sera raised in rabbits delayed the time of death of hamsters challenged with spores of C. difficile, but failed to protect animals from death (Table 2). In an active vaccination regimen, an extract of the two Slp proteins administrated with different adjuvants elicited an antibody response that partially protected hamsters from lethal challenge in the antibiotic-associated colitis model.
Another quite well conserved component of the cell wall of C. difficile, Cwp84, has recently been identified as a protease that degrades host tissues and may thus play a role in dissemination of the infection. Interestingly, anti-Cwp84 antibodies are found in patients suffering from CDI, showing that the protein is immunogenic. Immunization of hamsters with purified Cwp84 via different routes partially protected animals from colitis and death upon C. difficile challenge (Table 2). Cwp84 was encapsulated into pectin beads and the mucosal immune response tested in a hamster model upon oral administration. Approximately half of the animals survived a lethal challenge. However, no adjuvant was coencapsulated, and encapsulation was only tested with pectin beads, suggesting that more investigations with different beads and with codelivery of adjuvant may lead to the development of a more effective vaccine candidate.
Oligosaccharides found on bacterial cell surfaces also represent promising targets for antibacterial vaccine designs. Polyvalent carbohydrate structures usually induce a T cell-independent immune response resulting in IgM secretion. Conjugation of the carbohydrate antigen to a carrier protein is required for the induction of high titers of IgG antibodies because carrier-specific T helper cells induce antibody class switching, somatic hypermutations and the development of memory B cells. These antibodies enhance opsonization and killing of the bacteria by phagocytic cells. Unfortunately, these carbohydrates are usually highly strain specific. Glycoconjugate targeting for example, Haemophilus influenzae Type b, Streptococcus pneumoniae and Salmonella typhi are already licensed. Traditionally, production of these vaccines relies on purified capsular polysaccharides, but, due to advances in carbohydrate synthesis, production processes of carbohydrate-based vaccines with well-defined structures and circumventing fermentation and isolation processes are now developed. Recently, the structural characterization of the cell wall of C. difficile led to the identification of two capsular polysaccharides, PS-I and PS-II, the latter being found on hypervirulent B1/NAP1/027 and other strains. Two groups reported simultaneously the chemical synthesis of PS-II. In addition, Oberli et al. conjugated the carbohydrate to a carrier protein consisting of a nontoxic version of diphtheria toxin already used in several vaccines (variant CRM197). Upon immunization of mice with this conjugate vaccine, PS-II-specific IgG antibodies were produced (Table 2). In addition, anti-PS-II IgA antibodies were found in the stools of patients diagnosed with CDI. Together, these results and the progress in chemical synthesis of complex carbohydrate structures may allow the rational design of carbohydrate-based vaccines against CDI. However, more work is still required to better characterize the expression of PS-II in the various pathogenic strains of C. difficile and the capacity of anti-PS-II antibodies to prevent clinical manifestations of the disease.