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Vaccines against pertussis have been used for more than a hundred years. This review describes the development of whole-cell (wP) and acellular pertussis (aP) vaccines, which, as DTP combination vaccines, have significantly reduced morbidity and mortality from pertussis, and which currently serve as the building blocks for a variety of vaccines used to immunize all infants worldwide. Two series of efficacy trials done in the 1950s for wP vaccines and in the 1990s for aP-vaccines have established standards for studying vaccine effectiveness. However, irrespective of their longtime use, critical aspects of pertussis vaccines remain unknown, including the exact mechanisms of protective immunity and a correlate of protection. Research to improve on the effectiveness and the duration of protection is ongoing, but although the vaccines are not perfect, only their continued use with a high coverage will ensure that infants and children are being protected from pertussis.

Long-term stability is a desired characteristic of vaccines, especially anthrax vaccines, which must be stockpiled for large-scale use in an emergency situation; however, spontaneous deamidation of purified vaccine antigens has the potential to adversely affect vaccine immunogenicity over time. In order to explore whether spontaneous deamidation of recombinant protective antigen (rPA)—the major component of new-generation anthrax vaccines—affects vaccine immunogenicity, we created a “genetically deamidated” form of rPA using site-directed mutagenesis to replace six deamidation-prone asparagine residues, at positions 408, 466, 537, 601, 713, and 719, with either aspartate, glutamine, or alanine residues. We found that the structure of the six-Asp mutant rPA was not significantly altered relative to that of the wild-type protein as assessed by circular dichroism (CD) spectroscopy and biological activity. In contrast, immunogenicity of aluminum-adjuvanted six-Asp mutant rPA, as measured by induction of toxin-neutralizing antibodies, was significantly lower than that of the corresponding wild-type rPA vaccine formulation. The six-Gln and six-Ala mutants also exhibited lower immunogenicity than the wild type. While the wild-type rPA vaccine formulation exhibited a high level of immunogenicity initially, its immunogenicity declined significantly upon storage at 25°C for 4 weeks. In contrast, the immunogenicity of the six-Asp mutant rPA vaccine formulation was low initially but did not change significantly upon storage. Taken together, results from this study suggest that spontaneous deamidation of asparagine residues predicted to occur during storage of rPA vaccines would adversely affect vaccine immunogenicity and therefore the storage life of vaccines.

New anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies.

Antibodies against the protective antigen (PA) component of anthrax toxin play an important role in protection against disease caused byBacillus anthracis. In this study, we examined defined combinations of PA-specific monoclonal antibodies for their ability to neutralize anthrax toxin in cell culture assays. We observed additive, synergistic, and antagonistic effects of the antibodies depending on the specific antibody combination examined and the specific assay used. Synergistic toxin-neutralizing antibody interactions were examined in more detail. We found that one mechanism that can lead to antibody synergy is the bridging of PA monomers by one antibody, with resultant bivalent binding of the second antibody. These results may aid in optimal design of new vaccines and antibody therapies against anthrax.

Different types of anthrax toxin neutralization assays have been utilized to measure the antibody levels elicited by anthrax vaccines in both nonclinical and clinical studies. In the present study, we sought to determine whether three commonly used toxin neutralization assays—J774A.1 cell-, RAW 264.7 cell-, and CHO cell-based assays—yield comparable estimates of neutralization activities for sera obtained after vaccination with anthrax vaccines composed of recombinant protective antigen (rPA). In order to compare the assays, sera were assayed alongside a common reference serum sample and the neutralization titers were expressed relative to the titer for the reference sample in each assay. Analysis of sera from rabbits immunized with multiple doses of the rPA vaccine showed that for later bleeds, the quantitative agreement between the assays was good; however, for early bleeds, some heterogeneity in relative neutralization estimates was observed. Analysis of serum samples from rabbits, nonhuman primates, and humans immunized with the rPA vaccine showed that the relative neutralization estimates obtained in the different assays agreed to various extents, depending on the species of origin of the sera examined. We identified differences in the magnitudes of the Fc receptor-mediated neutralization associated with the J774A.1 cell- and RAW 264.7 cell-based assays, which may account for some of the species dependence of the assays. The differences in the relative neutralization estimates among the assays were relatively small and were always less than 2.5-fold. However, because toxin neutralization assays will likely be used to establish the efficacies of new anthrax vaccines, our findings should be considered when assay outputs are interpreted.

In May 2009 the National Institute of Allergy and Infectious Diseases hosted a workshop on serologic assays that support vaccine efficacy evaluations. The meeting promoted exchange of ideas among investigators from varying disciplines who are working on anti-infectious agent vaccines at different stages of development. The presentations and discussions at the workshop illustrated the challenges common across various pathogens with recurring themes: (1) A thorough understanding of the science regarding the pathogen and the host response to disease and immunization is fundamental to assay selection. (2) The intended use of the immunoassay data must be clearly defined to ensure appropriate specificity, accuracy, and precision; a laboratory must also commit resources to assure data quality and reliability. (3) During vaccine development, an immunoassay may evolve with respect to quality, purpose, and degree of standardization, and, in some cases, must be changed or replaced as data are accumulated. (4) Collaboration on standardized reagents and methods, harmonization efforts, and multidisciplinary teams facilitate consistent generation of quality data. This report provides guidance for effective development and utilization of immunoassays based on the lessons learned from currently licensed vaccines. Investigators are encouraged to create additional opportunities for scientific exchange, noting that the discussed themes are relevant for immunoassays used for other purposes such as therapeutics and diagnostics.

Anthrax toxin neutralization assays are used to measure functional antibody levels elicited by anthrax vaccines in both preclinical and clinical studies. In this study, we investigated the magnitude and molecular nature of Fc gamma (Fcγ) receptor-dependent toxin neutralization observed in commonly used forms of the anthrax toxin neutralization assay. Significantly more Fcγ receptor-dependent neutralization was observed in the J774A.1 cell-based assay than in the RAW 264.7 cell-based assay, a finding that could be due to the larger numbers of Fcγ receptors that we found on J774A.1 cells by using flow cytometry. Thus, the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by the assay depends on the specific cell type utilized in the assay. Using Fcγ receptor blocking monoclonal antibodies, we found that at least three murine Fcγ receptor classes, IIB, III, and IV, can contribute to Fcγ receptor-dependent neutralization. When antibodies elicited by immunization of rabbits with protective-antigen-based anthrax vaccines were analyzed, we found that the magnitude of Fcγ receptor-dependent neutralization observed in the J774A.1 cell-based assay was dependent on the concentration of protective antigen utilized in the assay. Our results suggest that the characteristics of the antibodies analyzed in the assay (e.g., species of origin, isotype, and subclass), as well as the assay design (e.g., cell type and protective antigen concentration), could significantly influence the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by anthrax toxin neutralization assays. These findings should be considered when interpreting anthrax toxin neutralization assay output.

Enzyme-linked immunosorbent assay (ELISA) has been widely used to evaluate antibody responses to pertussis vaccination and infection. A common reference serum is essential for the standardization of these assays. However, no internationally recognized reference serum is available. At the request of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO), a set of four candidate international standards has been prepared. These candidate materials have been assessed for suitability and compared to the widely used U.S. reference pertussis antiserum (human) lot 3, lot 4, and lot 5 by 22 laboratories from 15 countries in an international collaborative study. Laboratories measured immunoglobulin G (IgG) and IgA antibodies to pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), and fimbriae (Fim2&3) using their established immunoassays. The results of this study showed each of the four candidates to be suitable as an international standard. With the agreement of the participants, a recommendation has been made to the ECBS that the candidate material coded 06/140 be established as the First International Standard for pertussis antiserum (human), with the following assigned international units (IU): IgG anti-PT, 335 IU/ampoule; IgA anti-PT, 65 IU/ampoule; IgG anti-FHA, 130 IU/ampoule; IgA anti-FHA, 65 IU/ampoule; IgG anti-PRN, 65 IU/ampoule; and IgA anti-PRN, 42 IU/ampoule. No formal units have been proposed for anti-Fim2&3 because most assays used a mixture of fimbrial antigens. In addition, the candidate material coded 06/142 has been proposed as a WHO working preparation for characterization of assay systems.

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.

Previous studies of pertussis (whooping cough) that have derived diagnostic cut-off points for pertussis antibody levels have assumed a single distribution for antibody levels and have used small sample sizes. In a recent study of 5409 serum samples from the Third National Health and Nutrition Examination Survey (NHANES III), a finite mixture model was developed to examine the distribution of immunoglobulin G (IgG) antibody levels against pertussis toxin (PT), an antigen specific to the Bordetella pertussis bacterium. The mixture model identified three component populations with antibody levels greater than the quantitative assay's lower limit of quantitation (LLQ) and included a point distribution located at or below the LLQ to account for the excess number of antibody values that fell below the LLQ. The mixture model analysis accounted for the NHANES III design. A cut-off point for anti-PT IgG levels was chosen to have a 99 per cent model specificity based on the two overlapping normal distributions assumed for the two component populations with the highest antibody levels. This cut-off point may have a higher diagnostic sensitivity for acute B. pertussis infection than other cut-off points derived by assuming a single distribution for antibody levels.

Increasing evidence that the currently available acellular pertussis vaccines are not providing optimal control of pertussis in the United States and many other countries has stimulated interest in improvements of the current vaccines and in the development of new vaccines. A better understanding of the limitations of the current vaccines and the basis for the pertussis resurgence is needed to design improved vaccines. This article outlines several alternate approaches and summarizes the challenges related to the development of new or modified vaccines.

Serologic data often have a wide range and commonly do not approximate a normal distribution. Means, medians, SDs, or other conventional numerical summaries of antibody data may not adequately or fully describe these complex data. The reverse cumulative distribution plot is a graphic tool that completely displays all the data, allows a rapid visual assessment of important details of the distribution, and simplifies comparison of distributions.

Objective. To compare prospectively the reactogenicity and immunogenicity of two licensed whole-cell pertussis vaccines. Methods. We conducted a prospective, randomized, double-blinded assessment of two licensed whole-cell pertussis vaccines with diphtheria and tetanus toxoids that were included in a multicenter trial evaluating 13 acellular pertussis vaccines. Infants were immunized at 2, 4, and 6 months of age with a single lot of Lederle (309 infants) or Massachusetts Public Health Biologic Laboratories (MPHBL; 94 infants) vaccine. Results. The group receiving the Lederle vaccine demonstrated significantly higher antibody titers to pertussis toxin by enzyme-linked immunosorbent assay (ELISA) and by the Chinese hamster ovary cell pertussis toxin neutralization assay, and to fimbrial antigens by ELISA, as well as higher mean agglutinin titers. In contrast, the group receiving the MPHBL vaccine demonstrated higher ELISA antibody levels to filamentous hemagglutinin and pertactin. Similar differences were observed in the proportions of vaccinees seroconverting to these antigens. Rates of systemic and local reactions were relatively low for both vaccines. Although the Lederle product had substantially lower reactogenicity in this study than previously reported for that vaccine, the MPHBL vaccine was significantly less reactogenic in nearly all clinical categories. Conclusion. The two whole-cell vaccines demonstrated statistically significant differences in postimmunization antibody levels to all six evaluated pertussis antigens. Whether these statistically significant differences in antibody levels have clinical relevance is not clear. Rates of nearly all local and systemic reactions were significantly lower among the MPHBL group than the Lederle group. Licensed whole-cell diphtheria-tetanus-pertussis vaccines produced by different manufacturers cannot be assumed to be similar in reactogenicity or immunogenecity.

Objective. To evaluate the effect of simultaneous Haemophilus influenzae type b conjugate (Hib) vaccination on the safety and immunogenicity of selected acellular (DTaP) and whole-cell (DTP) pertussis vaccines with diphtheria and tetanus toxoids combined. Methods. Enrollment of infants into a large multicenter study of the safety and immunogenicity of 13 DTaP and 2 DTP vaccines was partially completed when the first Hib vaccine, HbOC (Haemophilus b oligosaccharide conjugate vaccine), was licensed for use in infants. Thereafter, at each immunization most infants received HbOC simultaneously with DTaP (or DTP), administered in opposite thighs. Postvaccination geometric mean titers or concentrations (GMTs) of pertussis antibodies as measured by six different assays were compared pairwise among groups of infants receiving 0, 1, 2, or 3 simultaneous HbOC immunizations. The incidence of reactions was compared between infants who received only DTaP or DTP and those who received HbOC simultaneously. Results. Comparison of postvaccination GMTs was possible among groups of infants receiving different numbers of simultaneous immunizations for 10 of the 13 DTaP and both DTP vaccines. Increased HbOC exposure had no consistent dose-response effect on antibody titers for DTaP or DTP vaccines in any assay. Significant diferences between groups in postvaccination GMTs were observed with 4 DTaP vaccines in 1 to 2 assays each; the GMTs were higher with increasing HbOC exposure for 2 DTaP vaccines and lower for 2 others. There was no significant increase in reactions with simultaneous HbOC and DTaP immunization. Conclusions. Based on these retrospective analyses, there did not seem to be an interference in pertussis immunogenicity or alteration in reactogenicity associated with the simultaneous administration of HbOC and DTaP. These findings are encouraging with respect to the development of DTaP-Hib combination vaccines.

In an effort to improve the reliability and reproducibility of serological assays forBordetella pertussis, a collaborative study was conducted to compare four different sources of pertussis toxin (PT) as coating antigens in the immunoglobulin G (IgG) anti-PT enzyme-linked immunosorbent assay (ELISA). Four sources of PT were used as coating antigens in the IgG anti-PT ELISA in four different testing laboratories (labs A to D) to determine whether the different antigen preparations and different laboratories influenced assay results. A panel of 60 sera consisting of deidentified human specimens from previous vaccination trials of healthy adults and infants and clinical specimens from outbreak settings was tested. In the four laboratories, each sample was tested three times with the four PT antigens according to the standard coating optimization and IgG anti-PT ELISA testing procedures used in that laboratory. Differences among the antigens, as well as intra- and interlaboratory variability, were evaluated. Excellent agreement was observed with the test panel results among the four antigens within each laboratory. Concordance correlation coefficient (rc) measurements among the different antigens ranged from 0.99, 0.99 to 1.00, 1.00, and 0.97 to 1.00 for labs A to D, respectively. The comparisons between pairs of laboratories also indicated a high degree of concordance for each PT preparation, withrcmeasurements between 0.90 and 0.98, 0.93 and 0.99, 0.92 and 0.98, and 0.93 and 0.99 for antigens 1 to 4, respectively. Relatively minor differences in results were observed among laboratories or among antigens, suggesting that the four PT antigens are quite similar and could be considered for acceptance in harmonized immunoassays used for serodiagnosis or vaccine evaluation.

The licensed anthrax vaccine and many of the new anthrax vaccines being developed are based on protective antigen (PA), a nontoxic component of anthrax toxin. For this reason, an understanding of the immune response to PA vaccination is important. In this study, we examined the antibody response elicited by PA-based vaccines and identified the domains of PA that contribute to that response in humans as well as nonhuman primates (NHPs) and rabbits, animal species that will be used to generate efficacy data to support approval of new anthrax vaccines. To this end, we developed a competitive enzyme-linked immunosorbent assay (ELISA), using purified recombinant forms of intact PA and its individual domains. We found that PA-based vaccines elicited IgG antibodies to each of the four PA domains in all three species. We also developed a competitive toxin neutralization assay, which showed that rabbits, NHPs, and humans all have functional antibody populations that bind to domains 1, 3, and 4. While the domain specificities of the antibody responses elicited by PA-based vaccines were similar in humans, NHPs, and rabbits, competitive assays suggested that humans may have a more significant secondary population of IgG antibodies that bind to partially unfolded or incorrectly folded PA. These findings provide information that will be useful when linking animal protection data to humans via an antibody bridge to establish efficacy of new anthrax vaccines.

Adequately sensitive and specific methods to diagnose pertussis in adolescents and adults are not widely available. Currently, no Food and Drug Administration-approved diagnostic assays are available for the serodiagnosis of Bordetella pertussis . Since concentrations of B. pertussis -specific antibodies tend to be high during the later phases of disease, a simple, rapid, easily transferable serodiagnostic test was developed. This article describes test development, initial evaluation of a prototype kit enzyme-linked immunosorbent assay (ELISA) in an interlaboratory collaborative study, and analytical validation. The data presented here demonstrate that the kit met all prespecified criteria for precision, linearity, and accuracy for samples with anti-pertussis toxin (PT) immunoglobulin G (IgG) antibody concentrations in the range of 50 to 150 ELISA units (EU)/ml, the range believed to be most relevant for serodiagnosis. The assay met the precision and linearity criteria for a wider range, namely, from 50 to 200 EU/ml; however, the accuracy criterion was not met at 200 EU/ml. When the newly adopted World Health Organization International Standard for pertussis antiserum (human) reference reagent was used to evaluate accuracy, the accuracy criteria were met from 50 to 200 international units/ml. In conclusion, the IgG anti-PT ELISA met all assay validation parameters within the range considered most relevant for serodiagnosis. This ELISA was developed and analytically validated as a user-friendly kit that can be used in both qualitative and quantitative formats. The technology for producing the kit is transferable to public health laboratories.

The epidemiologic features and clinical spectrum of pertussis in the elderly are poorly understood. In October 1985, the Wisconsin Division of Health investigated an outbreak of pertussis in residents of a nursing home in rural Wisconsin. Clinical information and nasopharyngeal swab and acute- and convalescent-phase serum specimens were obtained from all consenting residents and employees. Of 105 residents, 38 (36.2%) were seropositive, including four who were culture-positive for Bordetella pertussis. Culture-positive residents (age range, 52-81 years) had cough lasting 43-54 days. Three of these residents had paroxysmal cough, and all four had cough that interrupted sleep; none of the residents had cough with apnea or vomiting, and all recovered without sequelae. Of six seropositive residents with clinical pertussis, five lived on the south wing of the facility. Of 104 employees, 8 (7.7%) were seropositive, but none were culture-positive for B. pertussis. The higher attack rate for residents and the clustering of clinical cases were consistent with ongoing transmission within the nursing home.

Immunization with the 69-kD outer membrane protein (OMP) of Bordetella pertussis protected neonatal mice against lethal respiratory challenge with B. pertussis 18323. Active immunization elicited a serum IgG anti-69-kD OMP response at the time of challenge, with IgG anti-69-kD OMP antibodies detected in bronchoalveolar lavage fluid after challenge. Intravenous administration of BPE8, a monoclonal IgG1 anti-69-kD OMP, also protected young mice against B. pertussis challenge. Intravenously injected BPE8 was detected in the lungs of mice at the time of aerosol challenge, suggesting that the presence of specific antibody in the lungs may mediate protection. Thus the 69-kD OMP of B. pertussis is a protective antigen in mice that elicits specific serum antibody that can transude to the lung. The 69-kD OMP was detected in a preparation of a Takeda acellular vaccine by immunoblot analysis and a serum antibody response to the 69-kD OMP was observed in 18-mo-old children boosted with this preparation of Japanese acellular vaccine. Our results demonstrate that the B. pertussis 69-kD OMP is a protective antigen in animals, is immunogenic in humans, and is present in a preparation of acellular pertussis vaccine that is widely used in Japan. These findings indicate that the 69-kD OMP should be seriously considered as a candidate for inclusion in new formulations of antigenically defined acellular pertussis vaccines.

Numerous reports have documented that serologic methods are much more sensitive than culture for the diagnosis of pertussis in adolescents and adults. However, a standardized serologic test for pertussis is not routinely available to most clinicians, and the serologic test levels or cutoff points correlated with diseases have not been determined. The goal of the present study was to examine the distribution of immunoglobulin G (IgG) levels against three Bordetella pertussis antigens (pertussis toxin [PT], filamentous hemagglutinin [FHA], and fimbria types 2 and 3 [FIM]) and to determine population-based antibody levels for the purpose of establishing such diagnostic cutoff points. Enzyme-linked immunosorbent assays (ELISAs) were performed with sera from >6,000 U.S. residents aged 6 to 49 years who participated in the Third National Health and Nutrition Examination Survey. Mixture models were developed to identify hypothesized exposure groups and establish diagnostic cutoffs. Quantifiable (>20 ELISA units/ml [EU]) anti-FHA and anti-FIM IgG antibodies were common (65 and 62% of individuals, respectively), but quantifiable anti-PT IgG antibodies were less frequent (16%). Given the distributions of antibody levels, an anti-PT IgG level of ≥94 EU was proposed as the diagnostic cutoff point. Application of this cutoff point to culture-confirmed illness in a prior study investigating cough illness yielded a high diagnostic sensitivity (80%) and specificity (93%). A standardized ELISA for anti-PT IgG with a single serum sample appears to be useful for the identification of recent B. pertussis infection in adolescents and adults with cough illness. The PT cutoff point will be further evaluated in prospective studies of confirmed B. pertussis infection.

We have derived the mathematical relationship between the coefficient of variation associated with repeated measurements from quantitative assays and the expected fraction of pairs of those measurements that differ by at least some given factor, i.e., the expected frequency of disparate results that are due to assay variability rather than true differences. Knowledge of this frequency helps determine what magnitudes of differences can be expected by chance alone when the particular coefficient of variation is in effect. This frequency is an operational index of variability in the sense that it indicates the probability of observing a particular disparity between two measurements under the assumption that they measure the same quantity. Thus the frequency or probability becomes the basis for assessing if an assay is sufficiently precise. This assessment also provides a standard for determining if two assay results for the same subject, separated by an intervention such as vaccination or infection, differ by more than expected from the variation of the assay, thus indicating an intervention effect. Data from an international collaborative study are used to illustrate the application of this proposed interpretation of the coefficient of variation, and they also provide support for the assumptions used in the mathematical derivation.

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).

Objective. To compare the safety and immunogenicity of 12 different acellular pertussis vaccines combined with diphtheria and tetanus toxoids (DTaP) with one licensed diphtheria, tetanus, and whole-cell pertussis vaccine (DTwP) as a fourth-dose booster in children who had previously received DTaP or DTwP primary vaccinations.Methods. Healthy 15- to 20-month-old children were enrolled at six National Institutes of Health Vaccine Treatment and Evaluation Units. All had been randomly assigned to receive three primary doses of DTaP or DTwP at 2, 4, and 6 months of age as part of an earlier National Institutes of Health multicenter trial of DTaP vaccines in the same Vaccine Treatment and Evaluation Units. Parents recorded the occurrence and magnitude of fever; irritability; and injection site redness, swelling, and pain for 3 days after vaccination. Sera obtained before and 1 month after the booster vaccination were analyzed for antibody to pertussis toxin (PT), filamentous hemagglutinin (FHA), fimbriae (FIM), and pertactin (PRN). Diphtheria and tetanus toxoid as well as PT neutralizing (Chinese hamster ovary cell) and whole-cell agglutinating antibodies were measured on a subset of sera.Results. A total of 1293 children contributed fourth-dose reaction data. Reactions were less frequent after DTaP than after DTwP. For children vaccinated with a fourth dose of DTaP, which was the same DTaP as received in the primary series, fever and injection site redness, swelling, and pain increased in prevalence compared with the third dose in the primary series. For children receiving DTaP as a fourth dose, injection site redness and swelling occurred more frequently in DTaP-primed than in DTwP-primed children. Variation in the occurrence of reactions among DTaP vaccines was observed.A total of 1160 paired pre- and postvaccination sera were available for analysis. Serum antibody concentrations before boosting were lower than those obtained 1 month after the primary immunization. After the fourth dose, significant increases in antibodies directed against the included antigens were observed for all vaccines; postbooster vaccination antibody titers differed significantly among the DTaP vaccines. For children primed and boosted with the same DTaP, antibody levels were not directly related to the quantity of antigen included for PT, FHA, and FIM; for PRN, there was a closer relationship. Some DTaP vaccines given as fourth-dose boosters elicited antibody to PRN or FIM in some vaccinees, although the DTaP vaccines were not reported to contain these antigens; these responses were observed more frequently in DTwP-primed children. Agglutinin antibody rises were observed in all groups immunized with four doses of a DTaP vaccine containing FHA or PRN, regardless of whether the vaccine included FIM. Diphtheria and tetanus antibody levels exceeded the presumed protective concentration (0.1 IU/mL for diphtheria and 0.01 IU/mL for tetanus) after the fourth dose for all vaccinees.Conclusion. Although differences were observed in reaction rates among the DTaP vaccines given as a fourth dose, the DTaP vaccines were, in general, associated with fewer adverse events than a US-licensed DTwP. For DTaP vaccines, fever; irritability; and injection site pain, redness, and swelling occurred more frequently after the fourth dose than after the third dose of the same vaccine in the primary series. No DTaP was consistently most or least reactogenic or immunogenic. Although serologic correlates of pertussis immunity are not defined, it is clear that most DTaP vaccines can stimulate comparable or higher serum antibody responses than DTwP for those antigens contained in the vaccine.

Acellular pertussis vaccines are being evaluated in multiple clinical studies, and human immunogenicity data will likely be pivotal in the appraisal of vaccine responses between populations and the responses to different vaccine combinations. Antibody response to pertussis antigens is also used in the diagnosis of pertussis. An international study was designed to assess the comparability of data generated in different laboratories by enzyme-linked immunosorbent assays (ELISAs). Thirty-three participating laboratories were asked to quantitate specific antibody to pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), or fimbrial proteins (FIM) in 21 samples. Samples were to be assayed in triplicate in five independent assays by each ELISA routinely performed in the laboratory to assess intra-assay, interassay, and population variability. The mean sample values were used to compare quantitative results among the laboratories. Thirteen of the 32 laboratories which submitted evaluable data for an assay to measure antibodies to PT, 12 of 30 laboratories with assays for FHA, 10 of 17 laboratories with assays for PRN, and 6 of 13 laboratories with assays for FIM maintained a coefficient of variation below 20% for 75% of the samples tested. Assays that measure antibodies to FIM appear to be less precise than the other assays. Precision varied among laboratories that used similar methods. The relative values of intra- and interassay variabilities were not consistent for a given assay within a laboratory, indicating that the sources of these variability components may be unrelated. Precision and agreement appeared less reliable for samples with low antibody levels. Ranking and regression analyses suggest that some laboratories generated comparable quantitative results, although direct comparison or combination of results from different laboratories remains difficult to support. Calibration to the U.S. Reference Pertussis Antisera appears to have been successful at standardizing the results in some laboratories. Statistical analyses are affected by assay precision and are not necessarily reliable sole predictors of biologically relevant differences in quantitative results. If results from different laboratories must be compared, appropriate studies of precision and quantitative agreement should be conducted to support the specific comparisons.

Publisher Summary This chapter describes Bordetella pertussis and discusses recent advances in the development of pertussis vaccines. Bordetella pertussis is the pathogen that causes the human respiratory disease known as whooping cough, or pertussis. It is a fastidious Gram-negative bacillus that has a particular tropism for the ciliated epithelium of the respiratory tract. Pertussis begins as a mild upper respiratory infection that generally lasts 7–10 days, with nonspecific symptoms, usually in the absence of fever. During this initial and most infectious stage of disease, known as the catarrhal stage, the organism can be cultured from the nasopharynx. The disease usually progresses to the paroxysmal stage, typified by episodes of paroxysmal coughing, which, in young children, may be accompanied by an inspiratory whoop although this is often absent in infants younger than six months of age. Paroxysmal episodes may be followed by vomiting. The chapter also discusses new pertussis vaccines by examining the properties of the current whole cell vaccine. There is little doubt that whole cell pertussis vaccine (WCPV) has controlled pertussis in the communities where it has been adequately prepared and used under recommended protocols.

The Center for Biologics Evaluation and Research within the U.S. Food and Drug Administration has prepared a new U.S. Standard Pertussis Vaccine. Whole cell pertussis vaccine concentrate was diluted in 5% (w/v) lactose and lyophilized. The preparation was tested for toxicity, sterility, heterogeneity and residual moisture. Based on data from an international collaborative study involving 11 laboratories, the potency was estimated in relation to the U.S. Master Standard Pertussis Vaccine, Lot 4 and the International Standard for Pertussis Vaccine, Lot 2. The potency of the preparation was defined to be 90 units per ampoule. When reconstituted and stored according to instructions, no significant change in potency was observed in the 14 days following reconstitution. This material was shown to be suitable for a pertussis vaccine standard and accordingly it was designated as U.S. Standard Pertussis Vaccine, Lot 11 on March 22, 1994.

BackgroundAcute malaria has been associated with a decreased antibody response to tetanus and diphtheria toxoids, meningococcal, salmonella, and Hib vaccines. Interest in giving malaria drug therapy and prevention at the time of childhood immunizations has increased greatly following recent trials of intermittent preventive therapy during infancy (IPTi), stimulating this re-analysis of unpublished data. The effect of malaria chemoprophylaxis on vaccine response was studied following administration of measles vaccines and diphtheria-tetanus-whole cell pertussis (DTP) vaccines.MethodsIn 1975, six villages divided into two groups of children ≤74 months of age from Burkina Faso, were assigned to receive amodiaquine hydrochloride chemoprophylaxis (CH+) every two weeks for seven months or no chemoprophylaxis (CH-). After five months, children in each group received either one dose of measles or two doses of DTP vaccines.ResultsFor recipients of the measles vaccine, the seroconversion rates in CH+ and CH- children, respectively, were 93% and 96% (P > 0.05). The seroresponse rates in CH+ and CH- children respectively, were 73% and 86% for diphtheria (P > 0.05) and 77% and 91% for tetanus toxoid (P > 0.05). In a subset analysis, in which only children who strictly adhered to chemoprophylaxis criteria were included, there were, likewise, no significant differences in seroconversion or seroresponse for measles, diphtheria, or tetanus vaccines (P > 0.05). While analysis for pertussis showed a 43% (CH+) and 67% (CH-) response (P < 0.05), analyses using logistic regression to control for sex, age, chemoprophylaxis, weight-for-height Z-score, and pre-vaccination geometric mean titer (GMT), demonstrated that chemoprophylaxis was not associated with a significantly different conversion rate following DTP and measles vaccines. Seven months of chemoprophylaxis decreased significantly the malaria IFA and ELISA GMTs in the CH+ group.ConclusionMalaria chemoprophylaxis prior to vaccination in malaria endemic settings did not improve or impair immunogenicity of DTP and measles vaccines. This is the first human study to look at the association between malaria chemoprophylaxis and the serologic response to whole-cell pertussis vaccine.

Background.Diphtheria and tetanus toxoid combined with acellular pertussis (DTaP) vaccines are less reactogenic than diphtheria and tetanus toxoid combined with whole cell pertussis (DTwP) vaccines. However, local reactions increase in rate and severity with each successive DTaP dose, and swelling of the entire injected limb has been reported after booster doses.Methods.We reviewed reports of swelling of the entire thigh or upper arm after the fourth and fifth dose, respectively, of DTaP vaccines administered in the National Institutes of Health multicenter comparative DTaP studies. Relationships were explored among reports of severe swelling, rates of other reactions, quantity of vaccine contents, and prevaccination and postvaccination antibody levels to pertussis toxin, tetanus toxin, and diphtheria toxin.Results.Entire thigh swelling was an unsolicited reaction reported in 20 (2%) of the 1015 children who received 4 consecutive doses of the same DTaP vaccine. The reaction was associated with 9 of the 12 DTaP vaccines evaluated. Although there were no reports of swelling of the entire upper arm in 121 children given a fifth dose of the same DTaP, 4 (2.7%) of 146 recipients of 5 doses of a mixed schedule of DTaP vaccines experienced such swelling. Rates of other reactions were higher in children with entire thigh swelling than in those without. Of the children with entire thigh swelling, 60% had local pain, and 60% had erythema. All swelling subsided spontaneously without sequelae. There was a significant linear association between the rates of entire thigh swelling after dose 4 and diphtheria toxoid content in the DTaP products. Lesser degrees of swelling (>50 mm but less than entire limb) correlated with pertussis toxoid content after dose 4 and aluminum content after dose 5. No relationship was established between levels of serum antibody to diphtheria, tetanus, or pertussis toxin and rates of swelling of the whole thigh.Conclusions.Booster doses of DTaP vaccines can cause entire limb swelling, which is usually associated with redness and pain. Our data suggest that this extensive swelling reaction may be more common with vaccines containing high diphtheria toxoid content.

The National Institutes of Health (NIH) Vaccine and Treatment Evaluation Units (VTEUs) Collaborative Study of a Fifth Sequential Dose of 6 DT Acellular Pertussis (DTaP) Vaccines Compared with One Whole Cell Pertussis (DTwP) Vaccine in 4 to 6 Year-Old Children † 889

Six NIH Vaccine Treatment and Evaluation Units (VTEUs) enrolled 1340 15- to 20-month-old children to receive one of 12 DTaP or a DTwP vaccine as a booster after they had received 3 primary doses of the same DTaP or a DTwP at 2, 4, and 6 months of age. Fever, irritability, and injection site redness, swelling, and pain was assessed for 3 days after vaccination. Pre- and post- sera were analyzed for antibody (Ab) to pertussis toxin (PT), filamentous hemagglutinin (FHA), fimbriae (FIM), pertactin (PRN), and diphtheria (D) and tetanus (T) toxoids. Pertussis toxin neutralization (CHO) and agglutinating antibodies (AGG) were also measured for a subset of children. Common reactions increased after the booster dose compared with the primary series for both DTaP and DTwP; however, DTaP vaccines produced fewer reactions than DTwP. Significant variation in the occurrence of reactions among DTaP groups was observed. Priming with DTaP or DTwP did not influence the rate of reactions to DTaP boosting. Significant increases in Abs directed against the included antigens were observed for all vaccines; post booster Ab titers differed among the DTaP and DTwP vaccines. For children primed and boosted with the same DTaP, Ab levels correlated poorly with the quantity of antigen included for PT, D and T toxoid; for FHA, PRN and FIM there was a closer correlation. Children primed with DTwP and boosted with DTaP had higher Ab responses to PT than children who were primed with DTaP, indicating enhanced DTwP priming. Some DTaP vaccines given as a fourth dose in DTwP primed children elicited Ab to pertussis components not reported to be in the vaccines; suggesting the probable presence of trace quantities of these components. Although the highest AGG titers were observed in children immunized with DTaP vaccines containing FIM, AGG Ab responses occurred for all vaccines containing FHA. Use of DTaP for boosters in 15- to 20-month-old children has revealed reaction rates and immunogenicity results generally consistant with those following the primary series; however moderate but measurable increases in reactogenicity and some unexpected immunogenicity results were observed. Spon: NIH/NIAID& the vaccine companies.

• Relationships Between Functional Assays and Enzyme Immunoassays as Measurements of Responses to Acellular and Whole-Cell Pertussis Vaccines (1995;96:595-600): In the introduction, "WCL" is used incorrectly in three instances as an abbreviation for whole-cell pertussis vaccines. On page 595 (15 lines from the bottom of the right-hand column), "WCL pertussis immunization" should have read "whole-cell pertussis immunization." Similarly, on page 596 (in lines 21 through 23 of the left-hand column), "WCL vaccines" twice should have read "whole-cell vaccines." All other references to WCL in this article mean the specific product listed in Table 1 on page 596.

In the September 1995 supplement, titled Report of the Nationwide Multicenter Acellular Pertussis Trial (1995;96:547-603), two articles contain errors: • Description and Evaluation of Serologic Assays Used in a Multicenter Trial of Acellular Pertussis Vaccines (1995;96:570-575): On page 571, the 14th line under the heading "ELISA" contains an incorrect percentage for Tween-20 in the serum incubation buffer. It should be 0.05% (vol/vol) Tween-20, not 0.5%, as printed.

When WBC264-9C cells are preincubated with pertussis toxin, chemotaxis is inhibited and ADP-ribosylation of a membrane protein with a subunit Mr 41,000 is observed. Both the inhibition of chemotaxis and the ADP-ribosylation by pertussis toxin display a similar time lag, temperature dependence, and pertussis toxin-concentration dependence. Although the inhibition of chemotaxis and the ADP-ribosylation of the membrane protein are qualitatively correlated, nearly complete inhibition of chemotaxis occurs when there is only partial ADP-ribosylation of the membrane protein. Pertussis toxin-catalyzed ADP-ribosylation of the Mr 41,000 protein in WBC264-9C membranes is stimulated by GDP, GTP, and to a lesser extent by GMP; the nonhydrolyzable GTP analog guanosine 5'-[beta, gamma-imido]triphosphate has no effect. WBC264-9C membranes have a high-affinity GTPase activity, which is partially inhibited in membranes from pertussis toxin-treated cells. Neither GTPase activity nor adenylate cyclase activity in membranes from WBC264-9C cells is affected by fMet-Leu-Phe, an attractant for these cells. Our results suggest that a guanine nucleotide binding protein may be involved in chemotaxis, but they do not indicate an involvement of adenylate cyclase.

The management of children with human immunodeficiency virus infection in whom changes in pulmonary status develop can be complex. They are susceptible to a broad range of infections, and the clinician must be aware of the multiple etiologic possibilities and appropriate diagnostic tests. This report presents a potential pathogen that should be added to the list of organisms that can cause gram-negative pneumonia in children with HIV infection and highlights a diagnostic pitfall.

We prospectively compared the ability of anti-venom and edrophonium (Tensilon) to improve paralytic symptoms in 8 patients envenomed by the Philippine cobra (Naja naja philippinensis). Twenty, 50 or 100 ml of Philippine cobra antivenom were administered in a double-blind fashion by constant intravenous infusion over 30 min. Even the largest dose of antivenom failed to produce marked improvement within 2 h, though enzyme-linked immunosorbent assays and neutralization tests demonstrated that it possessed high titres of anti-neurotoxin antibodies. Tensilon given at 2 h was significantly more effective than antivenom at increasing the duration of upward gaze (78 +/- 28 vs 43 +/- 26 sec, P less than 0.001), and either completely reversed or markedly decreased paralysis in every patient. The Tensilon test should be given to all patients with paralytic envenoming by cobras, and anticholinesterases administered to those with a positive response.

The Sezary syndrome is a frequently lethal disease characterized by circulating malignant cells of thymus-derived (T)-cell origin. The capacity of circulating malignant lymphocytes from patients with this syndrome to synthesize immunoglobulins and to function as helper or suppressor cells regulating immunoglobulin synthesis by bone marrow-derived (B) lymphocytes was determined. Peripheral blood lymphocytes from normal individuals had geometric mean immunoglobulin synthetic rates of 4,910 ng for IgM, 1,270 ng for IgA, and 1,625 ng for IgG per 2 X 10(6) cells in culture with pokeweed mitogen for 7 days. Purified normal B cells had geometric mean synthetic rates of 198 ng for IgM, 145 ng for IgA, and 102 ng for IgG. Leukemic cells from patients with the Sezary syndrome produced essentially no immunoglobulins. Adding normal T cells to normal B cells restored their immunoglobin producing capacity. Leukemic cells from four of five patients tested had a similar capacity to help immunoglobulin synthesis by purified normal B cells. Additionally, Sezary cells from one patient studied induced a nearly 10-fold increase in IgA synthesis by lymphocytes from a child with ataxia telangiectasia and selective IgA deficiency. Furthermore, these Sezary cells induced more than a 500-fold increase in IgG and IgA synthesis by lymphocytes from a child with Nezelof's syndrome. When Sezary cells were added to normal unfractionated lymphocytes, they did not suppress immunoglobulin biosynthesis. In addition, unlike the situation observed when large numbers of normal T cells were added to purified B cells, there was no depression of immunoglobulin synthesis at very high malignant T-cell to B-cell ratios. These data support the view that Sezary T cells do not express suppressor cell activity. The results presented in this paper suggest that neoplastic lymphocytes from the majority of patients with the Sezary syndrome originate from a subset of T cells programmed exclusively for helper-like interactions with B cells in their production of immunoglobulin molecules.