As control, sera from nonimmunized (NI) children (= 8) were used. in developing countries) and 400,000 deaths each year.1,2 It remains an important cause of disease in both very young infants and in adolescent and adult populations despite the routine use of vaccination programs.2,3 In fact, seems to be re-emerging, featuring a modified epidemiology with a high incidence in early infancy and rising incidences in older children and adults.4,5 Several explanations have been put forward for the resurgence of infection in vaccinated populations.6 An apparent increase in incidence may result from improved surveillance, changes in case definition, and better Dutogliptin diagnostic techniques.7 However, it seems unlikely that these factors solely explain all cases in which a dramatic rise in has been observed. Other factors that might affect the incidence of include demographic changes, waning vaccine-induced immunity, changes in vaccine quality and/or vaccine coverage, or a decrease in the vaccine efficacy due to antigenic differences between circulating isolates and vaccinal strains.8 These antigenic differences may arise from genetic selective pressure induced by long term vaccination, or through changes in vaccine manufacturing that affect the antigens expressed by the vaccinal strains.7,8 Several vaccine combinations have been used to improve the establishment of the immunization programs, to consolidate the use of polyvalent vaccines, and to increase the coverage of each vaccine.7 Acellular pertussis vaccines were proven to be efficient and safe, due to the lower reactogenicity; in several countries their use has been preferred in the vaccination instead of the whole-cell pertussis.9C11 Brazil has been producing diphtheria-tetanus-pertussis vaccine since 1953, and from 1980, the 137 strain, from the National Institutes of Health (Bethesda, MD), was chosen to be used as antigen for the production of the whole cell pertussis vaccine (DTwP).12 Recent studies indicate that both immunization and infection during childhood do not lead to a permanent immunity against and, as a consequence, older children and adults are the main reservoirs of the infection.4,13 The aim of this study was to evaluate the antibody response of the whole cell pertussis vaccine immunized Brazilian children to various strains and their virulence factors in different periods of time after completion of the immunization process. Materials and Methods Chemicals and reagents. Tween 20, bovine serum albumin (BSA), goat anti-human alkaline phosphatase (IgG-AP), and p-Nitrophenyl Phosphate (pNPP) were purchased from Sigma (St. Louis, MO). BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and nitroblue tetrazolium (NBT) were from Promega Corp. (Madison, WI). Purified pertussis toxin (PT), pertactin (PRN), and filamentous hemagglutinin (FHA) were kindly provided by Dr. Rino Rapuolli (CHIRON S.p.A Laboratory, Siena, Italy). Strains and growth conditions. The strains used in the experiments were: 21A1, isolated from nasopharyngeal aspirate from an infant hospitalized with whooping cough clinical symptoms;14 137, obtained from the National Institutes of Health (NIH) and used for the preparation of the whole-cell vaccine in Brazil; 143, obtained from NIH; and Tohama, the Japanese vaccine strain. Bacteria were grown at 35.5C for 24 hours on BordetCGengou agar plates15 supplemented with defibrinated sheep blood at 25% and subcultured in Stainer and Scholte medium.16 For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis, bacteria grown on BordetCGengou agar plates at 10 109/mL were centrifuged at 7,000 for 45 minutes at 4C and cell pellets washed three times in saline solution. Samples were prepared in Laemmli buffer and boiled for 10 minutes. Human serum samples. Children (= 96) from Rio Preto, S?o Paulo, Brazil, were immunized with three doses of the whole-cell diphtheria-tetanus-pertussis vaccine, produced by Butantan Institute (S?o Paulo, Brazil), using strain 137 from NIH Hepacam2 as immunogen. Serum samples were collected after 2 (= Dutogliptin 12), 4 (= 13), 6 (= 22), 12 (= 26), and 24 (= 23) Dutogliptin months after the third dose, the time when the children were aging from 6 months to 3 years. The samples were centrifuged and the sera separated and stored at ?20C. Control serum samples were obtained from non-immunized children aging from 6 months to 1-year-old (= 8). This protocol was approved by the local ethics committee and the parents of the involved children authorized their participation in the study. Enzyme linked immunosorbent assay (ELISA). Microtiter plates were coated with 100 L.