Menthyldichlorophosphate: A chiral derivatizing agent for symmetrical diols

Menthyldichlorophosphate reacts readily with a variety of C2-chiral and meso diols to yield phosphate esters that exhibit useful diastereomeric differences in the 31P NMR spectra. Meso and d,l diols are easily differentiated with this reagent

Development of a Novel Vaccine Containing Binary Toxin for the Prevention of Clostridium difficile Disease with Enhanced Efficacy against NAP1 Strains.

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile

Purified vaccine components.

<p>Three micrograms 5mTcdA (lane A, 308kD), 5mTcdB (lane B, 269kD), proCDTb (lane C, 94kD) and 4mCDTa (lane D, 48kD) were loaded on a 8–16% Tris-Glycine (Invitrogen, Carlsbad, CA). GelCode Blue Stain Reagent (Thermo Fisher Scientific, Waltham, MA) was then used to stain the gel.</p

Potency of recombinant and native toxins in a cytotoxicity assay and mouse toxicity model.

<p>Potency of recombinant and native toxins in a cytotoxicity assay and mouse toxicity model.</p

Efficacy and immunogenicity of bivalent vaccine in hamsters.

<p>Kaplan-Meier graphs show survival curves for hamsters that were immunized intramuscularly four times with the bivalent vaccine containing 10μg 5mTxdA and 10μg 5mTxdB adjuvanted with ISCOMATRIX^™ and AAHS or adjuvant alone. Following the final immunization, hamsters were bled prior to challenge with either (A) VPI10463 (707 cfu, p<0.0001) or (B) BI17 (470 cfu, p>0.05) spores. Statistical differences in survival curves were calculated using Mantel-Cox test. (C) Pooled serum samples (day 0, 21, 42, 63 and 77) from the BI17 challenged hamsters were tested in an <i>in vitro</i> assay against Vero cells to measure neutralizing antibody activity against TcdA and TcdB. The ED50 was calculated as the serum dose that reduced cytotoxicity by 50%. Similar titers were observed from hamsters in the VPI10463 challenge. (D) Individual serum samples from day 77 from the VPI10463 and BI17 challenged hamsters were also tested for neutralizing antibody activity against TcdA and TcdB. Comparison of neutralizing antibody titers was performed using unpaired, two tailed t-test. * = p<0.05, *** = p<0.001, ns = no significant difference.</p

Tetravalent vaccine generates high neutralizing antibody titers in rhesus macaques.

<p>Rhesus macaques (n = 5) were given 3 immunizations with the tetravalent vaccine formulated with either ISCOMATRIX™ or ISCOMATRIX^™ plus AAHS on d0, d7, and d30. Control animals were immunized on the same days with a formalin inactivated toxoid vaccine adjuvanted with Rehydragel. Serum samples were collected on days 0, 7, 21 and 45. Individual (Day 45) or pooled (days 0, 7 and 21) serum samples were preincubated with active toxin prior to being added to Vero cells. ED50 values indicate the serum dilution at which the area of the Vero cell monolayer is reduced by 50%. Strong neutralizing antibody titers were generated against (A) TcdA, (B) TcdB, and (C) binary toxin. Day 45 titers in these graphs represent the mean value of the individual neutralizing titers. Neutralizing antibody titers to D) TcdA, E) TcdB, and F) binary toxin were measured from individual rhesus macaque serum samples on day 45. Statistical analysis was performed using One-way ANOVA and Tukey multiple comparisons test. * = p<0.05, ns = no significant difference.</p

Recombinant toxins and vaccines used in this study.

<p>Recombinant toxins and vaccines used in this study.</p