Bacteria, E. coli (ATCC number: 14948) and S. aureus (ATCC number: 25935,) kindly provided by Dr. H. K. Dannelly of the Department of Life Sciences), were cultured in LB broth (Difco, Detroit, MI) at 37°C for 14 hours and harvested with phosphate buffered saline (PBS). Cells were washed in PBS by centrifugation at 500 × g for 10 min at 4°C and then suspended to the appropriate density in PBS. Bacteria were killed by heating suspensions to 60°C for 1 hr.
Preparation of immunogen
Test vaccines consisted of either 5 × 106 CFU of E. coli or S. aureus and various adjuvants in a total volume of 400 μl. Adjuvants used in this study include: IFA (Sigma Chemical Co., St. Louis, MO), phytol (Pfaltz and Bauer Inc., Waterbury, CT) and PHIS-01 (Patent pending). PHIS-01 is one of several chemically modified phytol-based adjuvants developed in this laboratory.
Mice-immunization and challenge
Female BALB/c mice (6–8 weeks of age) were used for all experiments. These mice were bred and maintained at the animal care facility of Indiana State University. The use of these mice has been guided by strict adherence to an approved protocol prepared under the supervision and oversight of the Indiana State University Animal Care and Use Committee.
Immunization was carried out in groups of 4–5 mice using either 5 × 106 CFU of E. coli or S. aureus and test adjuvants (IFA, Phytol, or PHIS-01) in a total volume of 400 μL. Bacteria without adjuvant in a total volume of 400 μL PBS were used as control. Vaccines were administered intraperitoneally (I.P.) and animals were immunized three times at 10-day intervals. Sera were collected 5–7 days after each immunization for analysis by ELISA.
For the protection assay, mice were challenged with an IP injection of E. coli or S. aureus (106, 107, and 108 CFU/mouse) in 1.0 ml of PBS. Challenges took place on day 5 after the third immunization.
Preparation of bacterial cell lysates
Bacterial cultures were harvested and washed in PBS. Cells were lysed in 1 ml of buffer containing 8 M urea, 0.01 M Na-phosphate (dibasic), 0.01 M Tris-HCl (pH 8.0), and 5 μl of protease inhibitor cocktail. Cell debris was removed by centrifugation at 13,000 × g for 5 min at 4°C. Supernatant was collected and protein concentration was estimated from absorbance at 280 nm. Adopting a procedure reported by Gomez et al. , these lysates were used to coat 96-well microtiter plates for ELISA.
Enzyme-Linked Immunosorbent Assay (ELISA)
Antibody levels of mouse sera were measured routinely by a binding assay to antigen-coated ELISA plates:
(a) Antibody specific to bacterial strain: Cell-lysate coated ELISA plates were prepared by incubating polyvinyl 96-well plates with 10 μg/ml cell lysates in 0.01 M sodium bicarbonate solution overnight at 4°C. After blocking with 1% BSA/PBS overnight at 4°C, serially diluted sera obtained from immunized mice were added to each well, and incubated for 1 hr at 37°C. Plates were incubated with rabbit anti-mouse Ig-HRP and washed. Bound rabbit anti-mouse Ig-HRP was detected by addition of o-phenylene diamine (OPD), and the intensity was measured at 490 nm. Specific IgG antibodies were expressed as the mean ± SEM.
(b) Antibody specific to LPS: LPS (Sigma Chemical Co., St. Louis, MO) suspended in PBS were placed in poly-L-lysine (Sigma, St. Louis, MO) precoated ELISA plates and incubated for 1 hr at 37°C. The plates were washed three times with PBS containing 0.05% Triton-X, and ELISAs performed using conditions described by Takahashi et al .
To determine the characteristics of antibody response induced by vaccination, mouse immune sera were typed for IgM, IgG1, IgG2a, IgG2b, and IgG3 classes using anti-mouse Ig subclass-specific HRP-conjugated secondary antibodies (Zymed, San Francisco, CA), following the manufacturer's protocol. The ratio of IgG1 and IgG2a isotypes was calculated by dividing the A405 values for IgG1 by IgG2a.
SDS-PAGE and Western blot
Cell lysates, as described above, were mixed with an equal volume of SDS-PAGE sample buffer (Bio-Rad Laboratories, Hercules, CA) and electrophoresed on 12% polyacrylamide gels. The proteins were transferred to nitrocellulose membranes and rabbit anti-mouse Ig (A + M + G) (ICN, Irvine, CA) antibodiesand HRP-conjugated goat anti-rabbit Ig (Sigma Chemical Co., St. Louis, MO) were used to detect total Ig (M + G). HRP-conjugated rabbit anti-mouse IgG (Sigma Chemical Co., St. Louis, MO) was used to detect specific IgG type antibodies. Color development was accomplished using Supersignal® chemiluminescent (Pierce, Rockford, IL).
Proinflammatory cytokines, IL-6 and TNF-α levels in blood and peritoneal lavage were determined by ELISA. ELISA was performed in triplicate using specific mAbs (eBioscience, San Diego, CA) according to the manufacturer's instructions.
Monitoring of infection and mortality
These studies were conducted in PBS-treated control and experimental mice in various groups. These mice were vaccinated with killed bacterial lysates in the presence or absence of adjuvants. Animal mortality was assessed every 12 h during the first 3 days following bacterial challenges (106, 107 or 108 CFU/mouse) and peritoneal fluid and blood were cultured to confirm infection. Mortality occurred predominantly between 12 h and 36 h after challenge. At 18 h after bacterial challenge, blood and peritoneal lavages were obtained for quantitative culturing. Samples from all groups of mice were diluted in LB medium (1:200), and a 10 μl sample of each of these was streaked on agar plates using calibrated loops to detect bacteremia caused by >103 CFU/ml.
Statistical analyses of all data were done by the paired Student's t-test (Sigma Plot). For all statistical tests, alpha was set at 0.05. All data were expressed as mean ± SEM in the figures and tables.