Evaluation of a standardised Vi poly-l-lysine ELISA for serology of Vi capsular polysaccharide antibodies.

Typhoid vaccines based on protein-conjugated capsular Vi polysaccharide (TCVs) prevent typhoid in infants and young children. Analysis of the serum anti-Vi IgG response following immunisation against typhoid confirms the immunogenicity of TCVs and forms an important part of the pathway to licensing. Comparative studies could expedite the licencing process, and the availability of a standardised ELISA method alongside the 1st International Standard (IS) 16/138 for anti-typhoid capsular Vi polysaccharide IgG (human) will facilitate this process. To this end, a non-commercial ELISA based on a coat of Vi and poly-l-lysine (Vi-PLL ELISA) was evaluated by 10 laboratories. Eight serum samples, including IS 16/138, were tested in the standardised Vi-PLL ELISA (n = 10), a commercial Vi ELISA (n = 3) and a biotinylated Vi ELISA (n = 1). Valid estimates of potencies relative to IS 16/138 were obtained for all samples in the Vi-PLL ELISA and the commercial ELISA, with good repeatability and reproducibility evident from the study results and concordant estimates obtained by the two ELISA methods. The study demonstrates that the Vi-PLL ELISA can be used in clinical trial studies to determine the immunogenicity of TCVs.

The Spore Coat Protein CotE Facilitates Host Colonization by Clostridium difficile.

Clostridium difficile infection (CDI) is an important hospital-acquired infection resulting from the germination of spores in the intestine as a consequence of antibiotic-mediated dysbiosis of the gut microbiota. Key to this is CotE, a protein displayed on the spore surface and carrying 2 functional elements, an N-terminal peroxiredoxin and a C-terminal chitinase domain. Using isogenic mutants, we show in vitro and ex vivo that CotE enables binding of spores to mucus by direct interaction with mucin and contributes to its degradation. In animal models of CDI, we show that when CotE is absent, both colonization and virulence were markedly reduced. We demonstrate here that the attachment of spores to the intestine is essential in the development of CDI. Spores are usually regarded as biochemically dormant, but our findings demonstrate that rather than being simply agents of transmission and dissemination, spores directly contribute to the establishment and promotion of disease.

Mucosal Antibodies to the C Terminus of Toxin A Prevent Colonization of Clostridium difficile.

Mucosal immunity is considered important for protection against Clostridium difficile infection (CDI). We show that in hamsters immunized with Bacillus subtilis spores expressing a carboxy-terminal segment (TcdA26-39) of C. difficile toxin A, no colonization occurs in protected animals when challenged with C. difficile strain 630. In contrast, animals immunized with toxoids showed no protection and remained fully colonized. Along with neutralizing toxins, antibodies to TcdA26-39 (but not to toxoids), whether raised to the recombinant protein or to TcdA26-39 expressed on the B. subtilis spore surface, cross-react with a number of seemingly unrelated proteins expressed on the vegetative cell surface or spore coat of C. difficile These include two dehydrogenases, AdhE1 and LdhA, as well as the CdeC protein that is present on the spore. Anti-TcdA26-39 mucosal antibodies obtained following immunization with recombinant B. subtilis spores were able to reduce the adhesion of C. difficile to mucus-producing intestinal cells. This cross-reaction is intriguing yet important since it illustrates the importance of mucosal immunity for complete protection against CDI.

O-acetylation of typhoid capsular polysaccharide confers polysaccharide rigidity and immunodominance by masking additional epitopes.

In this work, we explore the effects of O-acetylation on the physical and immunological characteristics of the WHO International Standards of Vi polysaccharide (Vi) from both Citrobacter freundii and Salmonella enterica serovar Typhi. We find that, although structurally identical according to NMR, the two Vi standards have differences with respect to susceptibility to de-O-acetylation and viscosity in water. Vi standards from both species have equivalent mass and O-acetylation-dependent binding to a mouse monoclonal antibody and to anti-Vi polyclonal antisera, including the WHO International Standard for human anti-typhoid capsular Vi PS IgG. This study also confirms that human anti-Vi sera binds to completely de-O-acetylated Vi. Molecular dynamics simulations provide conformational rationales for the known effect of de-O-acetylation both on the viscosity and antigenicity of the Vi, demonstrating that de-O-acetylation has a very marked effect on the conformation and dynamic behavior of the Vi, changing the capsular polysaccharide from a rigid helix into a more flexible coil, as well as enhancing the strong interaction of the polysaccharide with sodium ions. Partial de-O-acetylation of Vi revealed hidden epitopes that were recognized by human and sheep anti-Vi PS immune sera. These findings have significance for the manufacture and evaluation of Vi vaccines.

Evaluation of the MTHFR A1298C variant in leukoaraiosis.

Vascular demyelinization of the white matter of the brain is referred to as leukoaraiosis (LA). This very frequent entity is associated with a cognitive decline, thereby resulting in a deteriorating quality of life. Besides poorly controlled hypertension and aging, its development is reported to be associated with an elevated serum homocysteine level. Although the methylenetetrahydrofolate reductase (MTHFR) C677T genetic variant is associated with an elevated serum homocysteine level, it has not been proved to be an independent risk factor for LA. The aim of the present study was to examine whether the MTHFR A1298C genetic variant, which is also believed to be unfavorable, is associated with the presence of LA. The clinical and genetic data on 198 LA patients and 235 neuroimaging alteration-free controls were analyzed. The presence of the A1298C or the 1298CC variant was calculated to be a risk factor for LA, as compared with the absence of both of them. The clustering of the heterozygous A1298C and C677T variants was proved to involve the risk of LA. Our results suggest that the MTHFR A1298C variant confers an independent genetic risk of LA, and this pathological role may be amplified by the MTHFR C677T variant.