International Bordetella pertussis assay standardization and harmonization meeting report. Centers for Disease Control and Prevention, Atlanta, Georgia, United States, 19-20 July 2007.

An international meeting on Bordetella pertussis assay standardization and harmonization was held at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, 19-20 July 2007. The goal of the meeting was to harmonize the immunoassays used for pertussis diagnostics and vaccine evaluation, as agreed upon by academic and government researchers, regulatory authorities, vaccine manufacturers, and the World Health Organization (WHO). The primary objectives were (1) to provide epidemiologic, laboratory, and statistical background for support of global harmonization; (2) to overview the current status of global epidemiology, pathogenesis and immunology of pertussis; (3) to develop a consensus opinion on existing gaps in understanding standardization of pertussis assays used for serodiagnosis and vaccine evaluation; and (4) to search for a multicenter process for addressing these priority gaps. Presentations and discussions by content experts addressed these objectives. A prioritized list of action items to improve standardization and harmonization of pertussis assays was identified during a group discussion at the end of the meeting. The major items included: (1) to identify a group that will organize, prepare, maintain, and distribute proficiency panels and key reagents such as reference and control sera; (2) to encourage the development and identification of one or more reference laboratories that can serve as an anchor and resource for other laboratories; (3) to define a performance-based assay method that can serve as a reference point for evaluating laboratory differences; (4) to develop guidance on quality of other reagents, e.g., pertussis toxin and other antigens, and methods to demonstrate their suitability; (5) to establish an international working group to harmonize the criteria to evaluate the results obtained on reference and proficiency panel sera; (6) to create an inventory to determine the amount of appropriate and well-characterized sera that are available globally to be used as bridging reagents for vaccine licensure; and (7) to seek specific guidance from regulatory authorities regarding the expectations and requirements for the licensure of new multicomponent pertussis vaccines.

Age-associated differences in immunoglobulin G1 (IgG1) and IgG2 subclass antibodies to pneumococcal polysaccharides following vaccination.

Immunoglobulin G (IgG) subclass antibody responses to pneumococcal vaccines were determined for human subjects in four age groups. The ratios of IgG1/IgG2 antibody concentrations declined with advancing age for all five of the serotypes tested. Protein-conjugate vaccines elicited enhanced IgG antibody responses over plain polysaccharide vaccines in infants but not in adult groups.

A randomized clinical trial of acellular pertussis vaccines in healthy adults: dose-response comparisons of 5 vaccines and implications for booster immunization.

The safety and immunogenicity of 5 acellular pertussis vaccines (ACVs) were compared in a multicenter, randomized, double-blind trial. A total of 481 healthy adults were given a single intramuscular booster dose of ACV or placebo. Three different dose levels were tested for 4 ACVs: full strength (the dose level proposed for infant immunization), one-third strength, and one-tenth strength. For 1 multicomponent vaccine, only the pertussis toxoid dose level varied. Minor injection site reactions were common and similar in frequency among vaccinated groups. Late-onset injection site reactions were seen in all ACV groups. Dose-related increases in mean antibody titers against vaccine antigens were seen after immunization with all ACVs. Antibody responses against antigens not known to be present in the vaccines were detected after immunization with 4/5 ACVs. Antibody levels fell significantly during the year after immunization. These data support evaluation of ACVs for broader use among adolescents and adults.

Characterization of human bactericidal antibodies to Bordetella pertussis.

The Bordetella pertussis BrkA protein protects against the bactericidal activity of complement and antibody; however, some individuals mount an immune response that overcomes this bacterial defense. To further characterize this process, the bactericidal activities of sera from 13 adults with different modes of exposure to B. pertussis (infected as adults, occupational exposure, immunized with an acellular vaccine, or no identified exposure) against a wild-type strain and a BrkA complement-sensitive mutant were evaluated. All of the sera killed the BrkA mutant, suggesting past exposure to B. pertussis or cross-reactive organisms. Several samples had no or minimal activity against the wild type. All of the sera collected from the infected and occupationally exposed individuals but not all of the sera from vaccinated individuals had bactericidal activity against the wild-type strain, suggesting that some types of exposure can induce an immune response that can overcome the BrkA resistance mechanism. Adsorbing serum with the wild-type strain removed the bactericidal antibodies; however, adsorbing the serum with a lipopolysaccharide (LPS) mutant or an avirulent (bvg mutant) strain did not always result in loss of bactericidal activity, suggesting that antibodies to either LPS or bvg-regulated proteins could be bactericidal. All the samples, including those that lacked bactericidal activity, contained antibodies that recognized the LPS of B. pertussis. Bactericidal activity correlated best with the presence of the immunoglobulin G3 (IgG3) antibodies to LPS, the IgG subtype that is most effective at fixing complement.

Reactogenicity and immunogenicity of a protein-conjugated pneumococcal oligosaccharide vaccine in older adults.

Healthy adults > or = 50 years old were immunized with either pentavalent Corynebacterium diphtheriae C7 (beta197) cross-reactive material (CRM197) protein-conjugated pneumococcal vaccine (CV) containing 10 microgram each of capsular oligosaccharides from serotypes 6B, 14, 18C, 19F, and 23F or with licensed (23-valent, 25 microgram/serotype) pneumococcal polysaccharide vaccine (PV). Adverse reactions, predominantly local in nature, occurred in 20 of 23 CV recipients versus 13 of 23 PV recipients (P<.05). Compared with mean postvaccination antibody concentrations in PV recipients, those induced by CV were not significantly different for serotypes 6B, 14, 18C, and 23F and were lower for 19F (P<.05). Six months later, reimmunization with PV of subjects who had initially received CV elicited a slight boost in antibody concentrations to levels that were not significantly higher than those achieved after the primary vaccination or than those in persons given a single dose of PV. Pneumococcal vaccines containing protein-conjugated oligosaccharides may offer no advantage over currently licensed preparations containing unconjugated polysaccharides for immunization of healthy older adults.

Relationships between functional assays and enzyme immunoassays as measurements of responses to acellular and whole-cell pertussis vaccines.

OBJECTIVE: To examine the relationships between functional assays and antigen-specific enzyme immunoassays in sera from a multicenter trial of 13 different experimental acellular pertussis vaccines and 2 licensed whole-cell vaccines, and to determine whether correlations previously observed among assays of specimens from pertussis patients and whole-cell vaccinees would apply to specimens from infants immunized with purified components in acellular vaccines. METHODS: Postimmunization sera were assayed for immunoglobulin G antibodies to pertussis toxin (PT), filamentous hemagglutinin, pertactin (PRN), and a mixture of types 2 and 3 fimbriae (FIM) by enzyme-linked immunosorbent assay, for whole-cell agglutinins (AGGs) and for PT-neutralizing antibodies by the Chinese hamster ovary (CHO) cell assay. Assay results were compared for individual sera, as well as for geometric mean antibody concentrations or titers (GMTs) calculated by vaccine or overall. RESULTS: For the 15 vaccines, the PT GMTs were highly correlated with the CHO assay GMTs (r = .92), and the FIM GMTs were highly correlated with the AGG GMTs (r = .96). For individual postvaccination sera, there was a significant correlation between the CHO titers and levels of antibody to PT whether the 15 vaccines were considered separately (.59 < or = r < or = .85) or combined (r = .81). For individual sera from infants immunized with the two whole-cell vaccines or any of the four acellular vaccines containing FIM, a strong correlation between AGG titer and FIM antibody was observed whether the vaccines were considered separately (.83 < or = r < or = .91) or together (r = .86). One vaccine without detectable FIM produced a measurable AGG response; for this vaccine, a moderate but significant correlation (R = .58) between PRN antibody and AGG titer was observed. CONCLUSION: These data indicate that appropriate antigen-specific enzyme-linked immunosorbent assays will furnish results similar to those provided by the CHO and AGG assays in the evaluation of the immunogenicity of component vaccines. Antibodies to FIM seem to include the most important AGGs; however, there is evidence that agglutination by PRN antibody may be detected in the absence of antibody to FIM.

Safety and immunogenicity of meningococcal A and C polysaccharide conjugate vaccine in adults.

A meningococcal vaccine containing group A and C polysaccharides conjugated to CRM197 was evaluated in 50 adults. Vaccinees were entered into one of five groups: 30 adults received a single dose of either 22, 11, or 5.5 micrograms of the conjugated A-C vaccine; 10 received an approved meningococcal vaccine; and 10 received saline injections. Local and systemic reactions to vaccines were recorded, and immune responses were determined. The experimental meningococcal vaccine was well tolerated, with the most frequent reaction being pain at the injection site. Both A and C polysaccharide components of the experimental vaccine were highly immunogenic, and total antibody concentrations 1 month postvaccination were not significantly different from the mean antibody concentrations among adults given the approved meningococcal vaccine. In addition, significant rises in immunoglobulin G, A, and M antibodies to both A and C polysaccharides occurred. Antibody concentrations measured at 6 and 12 months postvaccination had declined but remained significantly higher than prevaccination concentrations. Postvaccination meningococcal group C functional antibody activity increased more than 600-fold for both the polysaccharide and the conjugate vaccines. Further studies of this conjugated meningococcal vaccine are indicated for young children and infants.

Isotype and antigen specificity of pertussis agglutinins following whole-cell pertussis vaccination and infection with Bordetella pertussis.

Elevated agglutinin titers have been shown to correlate with protection from disease following whole-cell pertussis vaccination, but the isotype and antigen specificity of human agglutinating antibodies is unknown. In 13 immunoassays, immunoglobulin G antifimbria antibodies had the strongest correlation with agglutinin titers following culture-proven infection with Bordetella pertussis (R' = 0.79; P < 0.0001) and following whole-cell pertussis vaccination (R' = 0.87, P < 0.0001).

Acellular pertussis vaccines in infants: evaluation of single component and two-component products.

Two acellular pertussis vaccines, one containing only LPF toxoid (25 micrograms) and the other containing LPF toxoid (25 micrograms) and FHA (25 micrograms) and each combined with diphtheria and tetanus toxoids, were evaluated in two groups of 25 infants. A third group of 25 infants served as controls and received a DTP whole-cell pertussis vaccine. Infants given either acellular pertussis vaccine had significantly fewer local and systemic reactions than infants given whole-cell vaccine. Among the three vaccine groups, infants given the LPF vaccine (single component) had the highest concentration of antibody to LPF after three immunizations. Infants receiving the LPF/FHA vaccine (two-component) had the highest concentration of antibody to FHA after three immunizations. Infants vaccinated with the two-component vaccine had a significantly lower serological response to LPF than infants given the single component vaccine, as measured by either enzyme-linked immunosorbent assay or CHO cell assay. Further studies are necessary to determine why differences in immunogenicity of the two investigational vaccines occurred.

Safety and immunogenicity of Haemophilus influenzae type B-Neisseria meningitidis group B outer membrane protein complex conjugate vaccine mixed in the syringe with diphtheria-tetanus-pertussis vaccine in young Gambian infants.

To ensure compliance and to reduce costs it is important, especially in less developed countries, that programs of child immunization should require as few clinic attendances and as few injections as possible. Therefore we have investigated whether a Haemophilus influenzae type b conjugate vaccine could be given safely and effectively with diphtheria-tetanus-pertussis vaccine (DTP). One hundred twenty-six Gambian infants were given both polyribosylribitol phosphate (PRP)-outer membrane protein complex (PedvaxHIB) and DTP on the same day at 8, 12 and 16 weeks of age; 60 were given the vaccines mixed in the syringe and 66 were given the vaccines separately. To minimize the injection volume the dose of PRP-OMPC used in both groups was 7.5 micrograms, which is half the usual dose. There were no significant differences in anti-PRP antibody titers between the groups after 1, 2 or 3 doses. The geometric mean titers of antibody for the two groups combined were 0.29 micrograms/ml 1 month after the first dose, 1.03 micrograms/ml after the second dose and 1.11 micrograms/ml after the third dose. Concentrations of antibodies to diphtheria, tetanus and pertussis 1 month after the third dose were not significantly different between the two groups. Systemic side effects were reported with equal frequency in the two groups and were similar to those reported elsewhere for DTP. Tenderness at the injection site was more common where the combined injection (0.75 ml) had been given than where DTP alone (0.5 ml) had been given. The main drawback to the use of these 2 vaccines together is the complexity of the mixing procedure used in this clinical trial.

Severe reactions associated with diphtheria-tetanus-pertussis vaccine: detailed study of children with seizures, hypotonic-hyporesponsive episodes, high fevers, and persistent crying.

OBJECTIVE: The pathophysiology of severe reactions to diphtheria-tetanus-pertussis (DTP)vaccine is not well understood. Active pertussis toxin in DTP vaccine has been proposed to cause severe DTP vaccine reactions. Large doses of pertussis toxin cause hyperinsulinemia and hypoglycemia as well as leukocytosis with a predominant lymphocytosis in animal models. To learn more about the causes of and risk factors for severe DTP vaccine reactions, children experiencing severe DTP vaccine reactions were studied. DESIGN: Prospective, referral-based surveillance. SETTING: Los Angeles, CA. SUBJECTS: Children experiencing severe reactions within 48 hours of DTP immunization and evaluated within 24 hours of the reaction. Severe reactions included encephalopathy, persistent crying > or = 3 hours, hypotonic-hyporesponsive episodes (collapse episodes), fever > or = 40.5 degrees C, or seizures. Some comparisons were made between children with DTP vaccine-associated seizures and a comparison group of children experiencing febrile seizures unrelated to immunization. OUTCOME MEASURES: A history and physical examination were performed. Follow-up examinations were performed 1 month later. Blood was collected for complete blood cell count with leukocyte differential count, serum chemistry measurements, and insulin and glucose values. Serum was assayed for active pertussis toxin, both in free and immune-complex masked states. RESULTS: Sixty children experienced severe reactions within 48 hours of DTP immunization: 32 children had seizures only, 14 subjects had hypotonic-hyporesponsive episodes, 2 subjects had fever > or = 40.5 degrees C only, 4 subjects had persistent crying > or = 3 hours, 6 children had seizures and fever > or = 40.5 degrees C, and 2 children had persistent crying and seizures. The children with seizures had a high rate of personal and family histories of seizures, and 90% had documented fevers (> or = 38 degrees C). Persistent crying was associated with painful local reactions. Effects that may have been due to vaccine pertussis toxin were not found. Lymphocytosis did not occur, nor did hypoglycemia. Some relatively elevated insulin values were noted; however, this finding was also noted in the comparison group of children experiencing febrile seizures unrelated to immunization. No biologically active pertussis toxin was found in the acute sera of children experiencing severe DTP vaccine reactions. CONCLUSIONS: Seizures associated with DTP vaccine have similar clinical characteristics as febrile seizures, and persistent crying is initiated by painful local reactions. Vaccine endotoxin is a cause of febrile DTP vaccine reactions. We found no evidence that DTP vaccine pertussis toxin plays a role in severe DTP vaccine reactions.

Age-dependent V region expression in the human antibody response to the Haemophilus influenzae type b polysaccharide.

The human L chain antibody repertoire specific for the Haemophilus influenzae type b (Hib) polysaccharide (PS) is composed of kappa I, kappa II, kappa III, kappa IV, and lambda L chain V regions, but the most commonly occurring VL is encoded by the unmutated V kappa II-A2 gene. To determine whether this VL repertoire is influenced by age, we used idiotypic probes to monitor V kappa II-A2, V lambda, and V kappa III usage in the antibody response to an Hib PS-protein conjugate vaccine. A single dose of a vaccine consisting of Hib PS coupled to an outer membrane protein complex of Neisseria meningitidis was administered. Adults (n = 35), 18-month-old infants (n = 35), and 2-month-old infants (n = 46), all with > or = 0.9 microgram/ml anti-Hib PS antibodies, were tested for VL region markers in postvaccination sera. V kappa III anti-Hib PS antibodies were not detected in any of the 2-month-old infants but were detected in 29% of the 18-month-old infants and 69% of the adults (p < 0.001). The lack of kappa III antibodies in 2-month-old infants could not be accounted for by lack of a kappa response, because kappa antibodies to Hib PS were present (> 0.15 microgram/ml) in 45% of these infants. Hibld-1, an idiotope expressed by anti-Hib PS antibodies having the kappa II-A2 V region, was present in postvaccination sera of 66% of the adults and 80% of the 18-month-old infants but was less frequent in the 2-month-old infants (35%, p < 0.001). In contrast, Hibld-2, which is an idiotope expressed by a subset of V lambda VII anti-Hib PS antibodies, was rare or infrequent in adults and 18-month-old infants (0% and 6%, respectively) but was present in 43% of 2-month-old infants (p < 0.001). Our findings demonstrate that dramatic changes in VL region utilization occur in the human antibody response to this Hib PS conjugate vaccine as a function of age. Because previous studies have shown that V region usage correlates with antibody fine specificity and avidity for Hib PS, these age-related differences in V region expression may affect the ability of vaccines to confer protective immunity at different ages.

A search for Bordetella pertussis infection in university students.

University students with persistent cough of greater than or equal to 6 days' duration were evaluated for evidence of infection with Bordetella pertussis. Of 130 students studied during a 30-month period, 34 (26%) were found to have evidence of recent infections with B. pertussis. Infection was identified by direct fluorescent antibody assay of a nasopharyngeal specimen in one student and serologically in 33 additional subjects. B. pertussis was not recovered on culture of nasopharyngeal specimens from any subjects. Students with B. pertussis infection were identified in seven of the eight 3-month periods in which students were enrolled during the 30-month investigation, suggesting an endemic rather than epidemic pattern of infection in this university population. Illnesses of students with pertussis were similar to the illnesses of students without pertussis. The findings in this study suggest that adult populations in which endemic illness occurs at a relatively constant rate may be the reservoirs for pertussis outbreaks in susceptible children. Immunization programs in the future will need to employ booster doses for adults if complete control of B. pertussis infection is our goal.

Comparison of acellular and whole-cell pertussis-component diphtheria-tetanus-pertussis vaccines in infants. The APDT Vaccine Study Group.

In a multicenter, double-blind, randomized, longitudinal study, 252 children received licensed Lederle diphtheria-tetanus toxoids and pertussis vaccine adsorbed (DTP) at 2, 4, and 6 months of age, and 245 children received a DTP vaccine with the Lederle/Takeda acellular pertussis component (APDT) at the same ages. Both groups of children received APDT vaccine at 18 months of age. After each of the first three immunizations, APDT vaccine recipients had fewer local and systemic reactions than did DTP vaccinees. Reactions after the 18-month APDT vaccination were minimal in severity regardless of the vaccine previously received. Antibody responses to lymphocytosis-promoting factor and agglutinogens were more pronounced in DTP recipients; however, APDT recipients had a better serologic response to filamentous hemagglutinin, and responses to the 69K protein were equivalent. This APDT vaccine produces fewer reactions than the standard whole-cell DTP vaccine. The protective significance of the serologic responses to the APDT vaccine is unknown, but the greater response to filamentous hemagglutinin and equivalent response to the 69K protein compared with those to DTP vaccine seem promising.

Pathophysiology of reactions associated with pertussis vaccine.

Many adverse clinical events occur after pertussis immunization in children, but the pathophysiology is not well understood. It has been suggested that some of these adverse events may be due to biologically-active LPF and endotoxin present in DTP vaccines. Fifty-six children were studied who experienced severe reactions (fever greater than or equal to 40.5 degrees C, seizures, persistent crying greater than or equal to 3 hours or hypotonic-hyporesponsive episodes) within 48 hr of DTP immunization. Leukocytosis with neutrophilia was noted acutely (after vaccination) compared to follow-up (approximately one month later). No changes in insulin or glucose values were noted. Utilizing the CHO cell assay, no biologically-active LPF was found in the acute sera of children who had DTP-associated seizures. We found no evidence that biologically-active LPF or altered insulin/glucose metabolism were related to severe DTP-associated reactions.

Comparison of an acellular pertussis-component diphtheria-tetanus-pertussis (DTP) vaccine with a whole-cell pertussis-component DTP vaccine in 17- to 24-month-old children, with measurement of 69-kilodalton outer membrane protein antibody.

Healthy 17- to 24-month-old children, previously immunized with three doses of whole-cell diphtheria-tetanus-pertussis (DTP) vaccine, were enrolled in a multi-center double-blind, randomized study comparing a DTP vaccine with an acellular pertussis-component (APDT) and a conventional whole-cell pertussis-component DTP vaccine. Thirty-eight children received APDT vaccine, and 37 children received DTP vaccine. APDT vaccine recipients had significantly less local pain and warmth than DTP vaccine recipients. Antibody responses to lymphocytosis-promoting factor were similar in the two groups. The APDT vaccine recipients had a higher IgG antibody response to filamentous hemagglutinin than the DTP vaccinees had. Equivalent agglutinin responses were seen in the two groups. The APDT vaccine recipients had a significantly better antibody re-enzyme-linked immunosorbent assay, than DTP vaccinees had 1 month and 1 year after immunization. This APDT vaccine was immunogenic and caused fewer local reactions than conventional DTP vaccine when administered as a fourth dose to 17- to 24-month-old children.

Metabolic and hematologic effects and immune complex formation related to pertussis immunization.

Selected metabolic, hematologic, and immunologic functions were evaluated in 3- to 6-mo-old Finnish infants who received whole-cell pertussis-component diphtheria and tetanus toxoids and pertussis vaccine, adsorbed (DTP) vaccine, and in 4- to 6-y-old Los Angeles children who received either a licensed DTP vaccine or an acellular pertussis component DTP vaccine. One d after immunization, there was an increase in total leukocytes and neutrophils and a decrease in lymphocytes in all vaccinees. In 4- to 6-y-old children the leukocytosis and neutrophilia were greater in recipients who received the standard DTP vaccine than in vaccinees who received an acellular pertussis component DTP vaccine. In infants there was an increase in the mean plasma insulin concentration but no change in the glucose concentration 24 h after immunization; no increase in the mean plasma insulin was noted in the 4- to 6-y-old children. Three 4- to 6-y-old vaccinees had higher circulating immune complex concentrations after immunization and two of these children had high clinical reaction scores. The etiology of adverse reactions after DTP immunization is multifactorial. In contrast with findings in animals, our findings do not demonstrate a clinically significant effect due to lymphocytosis-promoting factor on glucose metabolism in vaccinated children. Neutrophilia in vaccinees is probably due to endotoxin, and some reactions may be due to circulating immune complexes.