Table 1.
Recombinant toxins and vaccines used in this study.
Fig 1.
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.
Table 2.
Potency of recombinant and native toxins in a cytotoxicity assay and mouse toxicity model.
Fig 2.
Efficacy and immunogenicity of bivalent vaccine in hamsters.
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 in vitro 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.
Fig 3.
Survival curves for immunized hamsters following a lethal challenge with C. difficile VPI10463, BI17, or 8864 spores.
Hamsters were immunized intramuscularly four times with the bivalent vaccine, tetravalent vaccine, binary toxin vaccine, or adjuvant alone. The 8864 challenge study did not include the binary toxin vaccine arm. (A) Hamsters (n = 9 for bivalent and tetravalent vaccine, n = 5 binary toxin vaccine or adjuvant control) were challenged with a lethal dose of VPI10463 spores (1200 cfu). (B) Hamsters (n = 15 for bivalent and tetravalent vaccine, n = 10 adjuvant control) received a lethal dose of NAP1 strain BI17 spores (425cfu). (C) Hamsters (n = 10 for all groups) received a lethal dose of strain 8864 spores (85cfu). Statistical analysis of survival curves was performed using Mantel-Cox test. * = p<0.05, ** = p<0.01, *** = p<0.001, ns = no significant difference.
Fig 4.
Comparison of immunogenicity of the bivalent or tetravalent vaccine in hamster vaccinated.
Sera from immunized hamsters (Fig 3) were assayed for functional antibody titers to TcdA, TcdB and binary toxin in a cell based assay. The ED50 was calculated as the serum dose that reduced cytotoxicity by 50%. There was no significant difference in TcdA and TcdB neutralizing antibody titers at day 56 in (A) the VPI10463 challenge, (B) the BI17 challenge, and (C) the 8864 challenge. Data was analyzed by two tailed t-test. (D) Pooled serum samples from the BI17 challenged animals were used to measure neutralizing antibody responses to TcdA, TcdB and binary toxin at days 0, 7, 28, 42 and 56. (E) ELISA binding titers were measured in pooled sera from B17 challenged hamsters for TcdA and TcdB. (F) Strong neutralizing antibodies were also generated to binary toxin by the tetravalent and binary toxin vaccines in the VPI10463, BI17 and 8864 challenges at day 56. One way ANOVA and Tukey's multiple comparisons tests were used to compared binary toxin titers. (G) Pooled serum samples from the BI17 challenged animals were also tested for binary toxin binding antibodies. Similar kinetics were observed in the VPI10463 and 8864 studies for neutralizing and binding antibody titers. * = p<0.05, ** = p<0.01, *** = p<0.001, ns = no significant differences.
Fig 5.
Tetravalent vaccine generates high neutralizing antibody titers in rhesus macaques.
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.