Acellular pertussis vaccines

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AceUular Pertussis Vaccines Mark R. Schleiss, MD, and Karen Dahl, MD

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ince the first crude extracts of Bordetella pertussis were utilized as vaccines shortly after the identification of the causative agent of whooping cough in the early 20th century, pertussis vaccines have engendered considerable controversy. Although pertussis immunization has saved countless lives and prevented considerable morbidity, the trade-off has been the fear that this vaccine has generated in much of the lay public. Indeed, pertussis immunization has been much maligned, being blamed, generally wrongly so, for many cases of illness, brain damage, and even death. The National Vaccine Compensation Act of 1987 was established to compensate families of children who had vaccine-associated injuries, many of which were attributed to administration of pertussis vaccine. Anti-immunization activists often point to pertussis vaccine as an example of the potential dangers associated with vaccination. Fortunately, the recent development and licensure of safer vaccines, the acellular pertussis vaccines, has represented a major advance in immunization practice. In this article, we review the basic bacteriology of B pertussis, the clinical manifestations of infection, and the history of the development of pertussis vaccines. Currently available acellular vaccines will be compared, and future potential developments in pertussis immunization, including prospects for combination vaccines, will be discussed.

Pertussis: Bacteriology and Molecular Microbiology The causative agent of pertussis is Bordetella pertussis, a small, gram-negative rod. The first successful From the Department of Pediatrics, Children's Hospital Medical Center, Cincinnati, Ohio. Dr Schleiss is an associate professor of Pediatrics in the Division of Infectious Diseases at the Children's Hospital Research Foundation in Cincinnati, Ohio. Dr DaN is a fellow in Pediatric Infectious Diseases in the Division of Infectious Diseases at the University of Colorado in Denver, Colorado. Curt Probl Pediatr 2000;30:185-201. Copyright © 2000 by Mosby, Inc. 0045-9380/2000/$12.00 + .15 5 3 / 1 / 1 0 7 8 3 4 d oi: 10.1067/m ps. 2000.107834

Curr Probl Pediatr, July 2000

recovery of this organism in culture was reported by Bordet and Gengou in 1906.1 The organism is fastidious, and difficult to grow in culture, a feature which not only complicates diagnosis of the illness, but has also hindered progress in identifying determinants of pathogenesis. In the Bordetella genus, there are 2 highly related organisms of clinical importance: B parapertussis, and B bronchiseptica. B parapertussis causes a pertussis-like syndrome in humans, which may be indistinguishable from that caused by B pertussis, although the illness caused by B parapertussis is usually milder.~ B bronchiseptica is only rarely a cause of respiratory illness in humans, although it is a common cause of respiratory illness in domestic animals. 3 Considerable knowledge has been gained in recent years about the molecular biology of B pertussis and related pathogenic species. Interestingly, molecular phylogenetic analyses have suggested that B pertussis, B parapertussis, and B bronchiseptica are highly interrelated, and DNA hybridization evidence suggests that these species may in fact represent subspecies of the same bacterium. 4 Currently, the complete DNA sequence of an isolate of B pertussis (Tohama strain 5) is being determined. This information, once available, should provide considerable insight into the molecular pathogenesis of infection. Molecular epidemiologic analysis suggests that clinical isolates may be quite variable. Recent evidence suggests a great degree of plasticity in B pertussis genome structure in clinical isolates, even in those from similar geographic locations, and extensive divergence of gene order appears to be a common occurrence, due to chromosomal inversions. 6,7 Other studies have shown extensive molecular divergence over time in the DNA sequence of immunogenic pertussis gene products such as pertussis toxin and pertactin (as described later). The extent to which this genomic plasticity complicates the development of protective immunity either after natural disease or vaccination is unknown. It may be that divergence of sequence in the genes encoding the major immunogenic targets of protective immunity, a form of "antigenic drift," is in part responsible for the

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Virulence Factors Important in Pathogenesis of Pertussis

I Pertussis Toxin

ADP-ribosyltransferase

• • • •

G-protein activation Lymphocytosis promotion T-ceil mitogen

Filamentous Hemagglutinin

• •

Adherence/binding to ciliated respiratory epithelium Hemagglutination activity

Pertactin

•

Outer membrane protein involved in adherence to

respiratory epithelial cells Cell invasiveness? Fimbrial Agglutinogens

•

Cell envelope proteins involved in attachment to respiratory epithelium Toxin with hemolysin and catalytic activity Inhibition of chemotaxis, phagocytosis and

Adenylate Cyclase

bacterial killing Endotoxin

•

Lipopolysaccharide toxin

Tracheal Cytotoxin

• •

Peptidoglycan cell well fragment Interacts with endotoxin to induce iNOS leading to nitric oxide-mediated destruction of respiratory

epithelium II

I

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1:1131. Virulence factors of B pertussis important in whooping cough disease pathogenesis and in protective immunity and vaccine design.

persistence of pertussis even among immunized populations. However, in spite of the need for additional information about the molecular biology of B pertussis, several key gene products have been well characterized (Fig 1). Knowledge of the role these gene products play in pathogenesis and protective immunity is essential for understanding the basis by which pertussis vaccines confer protection against disease.

Pertussis Toxin Arguably the most important component of the pertussis organism, both in terms of contribution to pathogenesis of disease as well as induction of immunity, is pertussis toxin (PT). 8 Pertussis toxin, also known as lymphocytosis-promoting factor, is an oligomeric protein made up of 5 subunits. The genes responsible for encoding the subunits of PT have been cloned and sequenced,9,1° All 5 subunits are coded by closely linked cistrons, The toxin is expressed through a polycistronic messenger RNA, with the order of the cistrons being S1, $2, $4, $5, and $3. The calculated molecular weights of the mature subunits are 26,024 kDa for S1; 21,924 kDa for $2; 21,873 kDa for $3; 12,058

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kDa for $4; and 11,013 kDa for $5. The holotoxin is comprised of 6 subunits, with the S 1 component designated as the A protomer. The B oligomer consists of 5 monomers, with the $5 oligomer functioning to link a $2-$4 dimer with a $4-$3 dimer. Each of the 5 subunits is secreted into the periplasm of B pertussis, where the 5 subunits are assembled into the oligomer, followed by release into the culture medium. In the absence of subunit $3, the remaining subunits are not secreted into the medium, thus suggesting that the assembled structure is necessary for toxin release. The mechanisms by which PT elicits disease remain poorly understood, but the identification of the crystal structure of PT, as well as observations from multiple in vitro studies in many laboratories, have begun to provide insights into structure-function relationships.l~ 14 PT is a member of the family of ADP-ribosyltransferases; enzymes which, through modification of host proteins, induce a variety of disease states. One of the most important modifications induced by PT is the ADP-ribosylation of host cell G-proteins, proteins involved in a variety of signal transduction pathways. Interestingly, the introduction of 2 amino acid substi-

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tutions within the enzymatically active subunit S 1 of PT abolishes its ADP-ribosyltransferase activity. This genetic alteration, while effectively "detoxifying" the protein, has no effect on the immunogenicity of PT, and has been exploited in acellular pertussis vaccine design.IS Other functions of PT include facilitation of attachment of B pertussis organisms to ciliated respiratory epithelium, promotion of lymphocytosis, stimulation of pancreatic islet cells, and sensitization to histamine. The B oligomer of PT also functions as a Tcell mitogen and contains hemagglutination activity. Although it has proven difficult to demonstrate the direct effect of PT outside of the respiratory tract, it is nonetheless plausible that PT plays a role in many of the systemic and, possibly, neurologic syndromes associated with pertussis. PT is highly immunogenic, and antibodies to PT are protective against both intracerebral and aerosol challenge in animal models of pertussis. 16,17Antibodies to PT are also associated with clinical immunity in humans, and it has been proposed that antibody to PT may be the single most important predictor of protection against disease. ~8,19All licensed acellular pertussis vaccines contain PT, and one vaccine contains only PT. However, in spite of multiple lines of evidence suggesting the importance of PT in immunity, several observations raise the question as to whether PT is of foremost importance in protection against disease. It is known that B parapertussis can cause a syndrome identical to that of pertussis, but B parapertussis does not express a functional PT, because of mutations in the promoter rendering the gene transcriptionally inactive. 2°,21 Although the coughing illness induced by B parapertussis is generally milder than that caused by B pertussis, clearly PT is not required for clinical illness. Further uncertainties about the essential role of PT in inducing illness stem from a clinical trial studying insulin response after glucose administration, in which humans were inoculated intravenously with PT. Although PT had an effect on glucosestimulated insulin secretion, subjects did not suffer any symptoms compatible with pertussis. 22 Thus, it appears that in spite of being a potent toxin with multiple biological effects, PT is neither necessary nor sufficient to induce the full clinical syndrome of pertussis.

Filamentous Hemagglutinin Filamentous hemagglutinin (FHHA)is a surface protein involved in binding of B pertussis to ciliated respira-

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tory epithelial cells. The filamentous hemagglutinin structural gene, fhaB, is translated into a precursor polypeptide, which then undergoes additional proteolytic processing to generate the mature 220-kd FHA product. 23,24An Arg-Gly-Asp (RGD) tripeptide found within the FHA coding sequence is necessary to confer its adherence properties. An internal in-frame deletion in fhaB, encompassing the RGD region, causes loss of B pertussis-binding to ciliated eukaryotic cells, confirming a potential role for this protein in host-cell infection. Adherence of B pertussis to monocytes/ macrophage cells also occurs by means of FHA through an interaction with the leukocyte integrin, complement receptor type 3 (CR3). 25 The FHA protein is highly immunogenic, both after natural infection and immunization with FHA-containing vaccines, and a number of immunodominant epitopes of the FHA molecule have been defined. 26 Mice immunized with FHA are protected against respiratory challenge with B pertussis, although interestingly, in contrast to PT-immunized mice, FHA-immunized mice are not protected against intracerebral challenge. 17 Intranasal immunization of mice with FHA has also been shown to confer protection against subsequent respiratory challenge, with pulmonary clearance correlating most strongly with the local antibody response. Most acellular pertussis vaccines contain FHA, although the amount of FHA present varies from vaccine to vaccine. Some epidemiological surveys suggest that the titer of anti-FHA antibody correlates with protection against disease. 27

Pertactin Pertactin (PRN) is a 69-kd protein present on the outer cell membrane of B pertussis. Analysis of the DNA sequence of the PRN gene identifies an open reading frame encoding a protein of 910 amino acids with a predicted M r of 93,478, implying that the 69-kd species is a processed form of a larger precursor. 28 This outer membrane protein is involved in adherence to respiratory epithelial cells. As with FHA, PRN contains an RGD integrin binding motif. The carboxy-terminal region of PRN contains a novel proline-glutamic acidproline repeat motif loop, which contains the major immunoprotective epitope. 29 The RGD motif is required for binding to respiratory epithelial cells. In addition to binding, PRN also appears to be able to elicit the phenotype of cell invasiveness. Interestingly, Salmonella strains engineered to express PRN had significantly increased invasiveness of mammalian cells

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compared with parental strains. 3° However, the role of invasiveness in the pathogenesis of disease is unclear, since B pertussis rarely (if ever) invades beyond the epithelial layers of the respiratory tract. PRN is highly immunogenic, and antibodies are found after natural infection and immunization with PRN-containing vaccines. The importance of humoral immune responses to PRN in protection against disease is unclear, and somewhat controversial. Clinical efficacy trials comparing acellular pertussis vaccines suggested increased efficacy of a 3-component vaccine containing PRN over a 2-component vaccine (as described later). However, other studies have identified the presence of antibodies to PRN in children who had neither a history of clinical pertussis nor antibodies against PT or FHA, raising concerns about the specificity of currently available assays for PRN antibodies. 31 Thus, although PRN clearly plays important roles in pathogenesis and immunity to B pertussis, it may be premature to conclude that antibody to PRN is essential for optimal protection against disease.

Fimbrial Agglutinogens Fimbrial agglutinogens are a complex family of proteins, which are found on the cell envelope of B pertussis. Although 8 different forms of agglutinogens are synthesized by B pertussis, only agglutinogens 1, 2 and 3 appear to be important in immunity. These proteins are involved in attachment of B pertussis to respiratory epithelial cells, although they are probably of lesser importance than FHA in this process. The fimbrial agglutinogens are highly immunogenic, and antibodies to these proteins are generally present both after immunization with whole-cell vaccine and after recovery from pertussis, although antibodies are difficult to demonstrate in young infants recovering from pertussis.32,33 Of the currently available acellular pertussis vaccines in the United States, only one contains timbrial agglutinogen proteins, although a World Health Organization (WHO) expert panel recommended that all whole-cell vaccines should contain agglutinogens 1, 2 and 3, underscoring the association between antibodies to these proteins with clinical immunity to pertussis. 34

Adenylate Cyclase Adenylate cyclase (AC) is a membrane-associated enzyme with both hemolysin and catalytic activity, which appears to be important in compromising the host phagocytic response to B pertussis infection. Mechanisms by which AC modulates immune response ap-

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pear to include inhibition of phagocytosis, chemotaxis, and bacterial killing, mediated through mechanisms including augmentation of production of cyclic adenosine monophosphate within the phagocyte. 35'36 AC is one of the few known protein toxins capable of penetrating directly into the cytosol of target cells across their cytoplasmic membrane without the need for endocytosis. This feature of AC has been exploited with recombinant AC toxoids, which have been recently used for delivery of CDS(+) T-cell epitopes into antigen-presenting cells in vivo, and for induction of protective antiviral, as well as therapeutic, antitumor cytotoxic T-cell responses. 37 Antibodies to AC are generated both after natural infection and immunization with whole-cell diphtheria-tetanus-pertussis vaccine (DTP). 38

Endotoxin and TrachealCytotoxin Like other gram negative bacteria, B pertussis produces endotoxin. The presence of endotoxin in wholecell pertussis vaccines has been proposed as one of the reasons for the reactogenicity of these products. Until recently, no specific pertussis-induced pathology could be linked to endotoxin. However, a novel role for endotoxin in pertussis-induced pathology has now been identified. Pertussis endotoxin interacts with another pertussis virulence factor, tracheal cytotoxin, a fragment released from the peptidoglycan of the pertussis cell wall during pertussis replication. This interaction activates production of nitric oxide (NO) through induction of NO synthase (iNOS). The induction of iNOS is specific to nonciliated, mucus-secreting cells in the respiratory tract. Diffusible NO produced by secretory cells may, in turn, be toxic to the neighboring ciliated cells. 39,4° This endotoxin/tracheal cytotoxin induction of NO may help to explain the extensive destruction of ciliated epithelial cells seen in severe whooping cough.

Pertussis: Epidemiology and Clinical Manifestations

Magnitude ,of Pertussis-RelatedDisease Burden Pertussis continues to be a major cause of morbidity and mortality worldwide. It has been estimated that 40 million cases occur annually, with 360,000 pertussisrelated deaths. 41 The worldwide incidence and age distribution of pertussis are affected by immunization

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FIG 2. Incidence of pe~tussis in United States by year, 1968-1998. Pertussis epidemics tend to occur in cyclic fashion every 3 years. Pertussis incidence reached a nadir in mid 1970s, but has been increasing steadily over past 15 years. 43'56 Figure reproduced from reference 43.

status, as well as socioeconomic factors. In the developing world, or in countries where immunization is not routinely practiced, pertussis is a disease predominantly of infants and young children. Unfortunately, this is the population at greatest risk for severe pertussis-related complications. Infants are more likely than older children to have pneumonia, encephalitis, or pertussis-related seizures. 42 The incidence of pertussisrelated mortality is also highest in this group. Infection in unimmunized populations appears to be inescapable, because of the extremely high transmission rate of pertussis. Pertussis is also an epidemic disease, with periodic cycles of every 3 to 4 years, irrespective of whether the population is immunized or not. In the United States, routine immunization programs, begun in the 1940s, have dramatically changed the incidence of disease but have not eradicated pertussis. Indeed, although pertussis declined dramatically between 1940 and the mid 1970s, with the number of reported cases reaching a nadir in 1976, more recently pertussis appears to be increasing in incidence (Fig 2). 43 The reasons for this apparent increase in pertussis incidence are not clear. Better surveillance and recognition of pertussis undoubtedly has played a role. More likely, the overall increase in pertussis cases reflects the substantial increases in the number of cases in ado-

Curr Probl Pediatr, July 2000

lescents and young adults. Increasingly, the population contains individuals with vaccine-derived immunity, and not disease-related immunity. Immunity in those who recover from whooping cough appears to be lifelong, but this is not true for vaccine-derived immunity. Consequently, as vaccine immunity wanes, young adults appear to be increasingly susceptible to acquisition of pertussis, and therefore represent an increasingly important reservoir for this infection. 44 Hence, for ultimate eradication of pertussis, adult immunization will almost certainly be necessary.

Clinical Manifestations of Pertussis A patient recently admitted to Children's Hospital in Cincinnati had a classic clinical course of pertussis. This 10-week-old infant was brought to the emergency department by her mother with the chief complaint of "difficulty in breathing." Her mother stated that the infant had been coughing for several weeks, but more recently the coughs had started to come in "fits" and seemed to have worsened over the past several days. Post-tussive emesis was noted, as well as decreased oral intake. The infant had been seen on several occasions by her pediatrician for these symptoms in the weeks preceding admission. At the time of admission, the infant was noted to be afebrile (temperature, 98.0°F) and was alert and play-

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Pertussis: Clinical Manifestations

Catarrhal

, Rhinorrhea, conjunctival injection • Highly contagious period (1-2 weeks)

Paroxysmal

• Forceful, paroxysmal cough with "whoop" • Morbidity high in infants due to cyanosis (2-6 weeks)

Convalescent

• Recovery phase Prolonged cough common (1-2 weeks, but cough may persist several months)

FIG 3. Clinical presentation of pertussis.Panel A, chest radiograph of infant with culture and DFA-confirmed pertussisrevealing diffuse infiltrates. Panel B, summary of clinical stages of pertussis (catarrhal, paroxysmal, and convalescent).

ful, although tachypneic, between episodes of spasmodic cough. Pulse was 170 bpm, and respiratory rate was 78 breaths per minute. Physical examination was remarkable for diffuse crackles heard throughout the lung fields. Suprasternal and subcostal retractions were present. Laboratory studies revealed an elevated peripheral white blood cell count (61,100 with 20% neutrophils, 64% lymphocytes, 7% monocytes, and 7% atypical lymphocytes). Chest radiograph revealed patchy infiltrates in the right upper lobe and bilateral lower lobes (Fig 3, panel A). A nasopharyngeal swab was obtained for pertussis DFA-testing, which was strongly positive, as was culture. During the course of the hospitalization, the coughing fits gradually decreased but did not completely resolve. Infiltrates on chest radiograph resolved gradually. The infant and her family were treated with 10 days of erythromycin. A 17-year-old cousin with a mild chronic cough was thought to be the index case. 190

This classic case of B pertussis pneumonia illustrates several points: the younger the infant, the higher the risk of a complicated infection; treatment in the paroxysmal stage is supportive; the index case is often a young adult with mild disease. After acquisition of pertussis, the average incubation period is approximately 7 to 10 days. Symptomatic illness then supervenes. Classically, clinical manifestations of pertussis are divided into 3 stages: the catarrhal phase, the paroxysmal phase, and the convalescent phase (Fig 3, panel B). 45 The catarrhal phase is extremely nonspecific. Symptoms may simply be those of an upper respiratory tract infection (ie, rhinorrhea, sneezing, conjunctival infection, and mild cough). Because the symptoms are nonspecific, the diagnosis of pertussis may not be suspected. However, a low index of suspicion is important, because patients are highly contagious during this phase, and the administration of appropriate antibiotics significantly decreases contaCurr Probl Pediatr, July 2000

gion. This stage persists for approximately 1 to 2 weeks. As the paroxysmal stage begins, the cough increases in frequency and intensity. Paroxysms, a rapid series of 10 or more forceful coughs, occur during expiration, and are followed by inspiratory effort against the narrowed glottis, leading to the classic "whoop" of whooping cough. These paroxysmal episodes may occur up to 30 times a day. Remarkably, children may look well between episodes. However, paroxysms may be associated with substantial morbidity, including cyanotic episodes. The paroxysmal phase ranges from 2 to 6 weeks. Antibiotic therapy has little effect on limiting the course of disease once this stage occurs. As the paroxysms of cough gradually subside, the convalescent stage follows, lasting for 1 to 2 weeks. Recovery is gradual, and cough may persist for several additional months.

Diagnosis and Management In a young child with classical whooping cough, the diagnosis can be virtually certain just on clinical grounds alone. However, ancillary laboratory studies are useful in confirming pertussis. The complete blood cell count often reveals an impressive lymphocytosis, with total white blood cell counts sometimes approaching 100,000 WBC/mL. Culture of B pertussis is confirmatory, but, as noted, the organism is fastidious and difficult to grow. For optimal yield, nasopharyngeal specimens should be inoculated onto appropriate culture media as quickly as possible. The yield by culture is highest during the catarrhal phase. Direct fluorescent antibody staining is a useful test, which complements culture, and should be performed in parallel with culture to maximize diagnostic yield. More recently, polymerase chain reaction testing has shown promise as an even more sensitive methodology for detecting B pertussis DNA sequences, 46 although this assay does not yet have widespread availability. In certain cases, serology may be warranted to attempt to establish the diagnosis of pertussis. Serologic assessment of previously immunized individuals may be fraught with difficulties in interpretation, since IgG antibodies may be present because of vaccine-derived immunity. Therefore, demonstrating a substantial rise in antibody titer by using paired (acute and convalescent) sera is ideal. Alternatively, measurement of pertussis-specific IgA responses can be performed, since IgA responses more often reflect actual infection, rather than vaccine-induced immunity.47,48

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Therapy It is important to keep in perspective what are reasonable expectations with regard to antibiotic treatment of pertussis. Treatment, in general, will only shorten the clinical course of the disease if begun during the catarrhal phase of the illness. However, antibiotics are indicated during all phases of the illness, to hasten clearance of the organism and limit contact spread. Erythromycin, particularly erythromycin estolate, has traditionally been considered the most useful antibiotic for permssis.49 However, strains of pertussis resistant to erythromycin have been described.5° Moreover, the recent linkage between the use of erythromycin in infants and the possibility of inducing pyloric stenosis has prompted consideration of other regimens.5a Newer macrolide antibiotics, such as clarithromycin and azithromycin, appear to be of therapeutic benefit against pertussis and represent reasonable alternatives. 52 If a macrolide antibiotic cannot be tolerated, trimethoprim-sulfamethoxazole is recommended. Although antibiotics are important in controlling the spread of pemlssis, the aggressive use of pertussis vaccines clearly plays a much more significant role in limiting the spread of this disease.

Whole-Cell Pertussis Vaccines

History of Whole-Cell Vaccines Shortly after the successful isolation in culture of B pertussis in 1906 by Bordet and Gengou, 1 attempts were made to develop pertussis vaccines. The first published report of the use of a pertussis vaccine was in 1912, and a pertussis vaccine was licensed for use in the United States in 1914. 53 These early vaccines were preparations consisting of suspensions of killed organisms, hence referred to as "whole-cell" vaccines. The earliest vaccines were extremely crude preparations, which contained mixed respiratory flora in addition to B pertussis. Initially, vaccine efforts were directed at therapy of established disease in addition to prevention of disease. The first clinical trials, which indicated the efficacy of pertussis vaccines, were performed by Madsen in 1923 and 1929, and published in 1933. 53 These studies, performed in the Faroe Islands, were performed by using killed whole-cell vaccine during epidemics of pertussis. The first study was a therapeutic study. A group of 450 individuals received 3 doses of vaccine, injected at 4 day intervals. Compared with an unimmunized control group of 405 patients, patients who received vaccines had milder

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disease and, significantly, dramatically reduced mortality (1 death compared with 13 deaths in the control group). In the second Madsen study, patients immunized with pertussis vaccine had a much lower attack rate than controls, and the disease was significantly milder.53 The encouraging results noted by Madsen were extended in a series of studies performed by Kendrick and Eldering in the 1930s and 1940s. 54 Important advances in the field during this period were the introduction of standardized culture conditions for preparation of vaccine material, improvements in ability to quantify the number of organisms in the vaccine preparation, and the recognition that use of fresh, rapidly growing organisms (so-called "phase 1" organisms) resulted in a safer, better-tolerated product. Vaccine potency could be quantified more precisely after the development of the "mouse protection test." In this test, mice are immunized with pertussis vaccine and then challenged by inoculation of live organisms intracerebrally. 55 Immunogenicity of the vaccine preparation is quantified by comparing mouse survival rates with survival rates from a "standard" vaccine preparation of defined potency. Although recent advances in the understanding of the host response to pertussis have made it clear that the host responses involved in protective immunity may differ greatly between mice and humans, this test nonetheless made it possible to quantify vaccine lot potency. Pertussis vaccines have been licensed and marketed since the mid 1940s, and have been standardized by federal regulation (chiefly by use of the mouse protection test) since 1953. Remarkably, the whole-cell vaccines, which have been produced, marketed, and used in clinical practice, have been essentially unchanged since that time. They are derived from B pertussis cultures grown in Bordet-Gengou media, inactivated with thimerosal, and adsorbed in combination with diphtheria and tetanus toxoids onto aluminum salts in order to enhance immunogenicity. The final product is therefore referred to as the "wholecell" diphtheria-tetanus-pertussis vaccine, or DTR

Efficacy of Whole-Cell Vaccines The introduction of whole-cell pertussis vaccines had a dramatic effect on the incidence, severity, and morbidity of pertussis in the human population. In the United States, the incidence of pertussis exceeded 300,000 cases annually in the 1940s, but after widespread introduction of permssis vaccines, a steady decline in cases of whooping cough has been observed, reaching a nadir of a few thousand cases annually in the 1970s. 56 In spite

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of the impressive success of pertussis vaccines, it remains unclear what the precise efficacy of whole-cell vaccine is in preventing disease. Vaccine efficacy estimates in the United States range from less than 70% to greater than 95%, depending upon the definition of whooping cough used in the particular study. Other factors that impact upon efficacy include the number of doses of vaccine administered, the population under study, and the prevalence of pertussis in the community under study. Some studies have also revealed substantial manufacturer-to-manufacturer variation in the immune responses elicited by the various whole-cell vaccines recently used in the United States. 57 These differences may have played a role in the surprisingly low efficacy observed for whole-cell vaccines when compared with acellular pertussis vaccines in the European trials, which will be discussed further ("Acellular Permssis Vaccines" section). However, there can be little question that the widespread use of whole-cell pertussis vaccine has been associated with remarkable declines in the incidence of whooping cough in developed countries. Compelling evidence of the benefits of whole-cell pertussis immunization in reducing pertussis disease comes from observations of the effects of withdrawal of vaccination in developed countries where vaccine use was suspended. In Japan, after over 2 decades of favorable experience with whole-cell DTR the death of 2 infants after vaccination led to the recommendation in 1975 by the Ministry of Health and Welfare that pertussis vaccine use be suspended. The number of cases of whooping cough in Japan rose dramatically, from 206 cases in 1971 to over 12,000 cases in 1979. 58 A similar experience was noted in Great Britain in the 1970s. Because of concerns about the safety of wholecell vaccines, immunization rates fell significantly, and a major epidemic of pertussis ensued. 59 Similar results were also observed in Sweden, when vaccination with whole-cell vaccine was suspended in 1979. 6o The resurgence of whooping cough in these diverse cultures stands as a testimonial to the stubborn persistence of B pertussis in the human population. Suspension of vaccine programs lead to significant pertussis-associated morbidity, and set the stage for the development and testing of the acellular pertussis vaccines.

Adverse EventsAssociated with Whole-Cell Vaccines The trade-off for the protection that whole-cell vaccines provided has been a troublesome reactogenicity

Curr Probl Pediatr, July 2000

profile. A harbinger of future problems associated with whole-cell pertussis vaccines was observed in the landmark Faroe Islands studies. 53 Although this trial clearly established the value of immunization against whooping cough, 2 deaths were noted in immunized infants after the second dose of vaccine. These deaths, attributed to "shock," may have been due to impurities in this crude, early vaccine preparation. Although such catastrophic adverse events are extraordinarily rare, the side effects of whole-cell pertussis vaccination have generated considerable fear, anxiety, and litigation in recent years, and were a major driving force in the development of acellular vaccines. Most reactions to whole-cell pertussis vaccines are fortunately minor. Redness and pain and swelling at the injection site occur in approximately half of DTP recipients. Mild systemic reactions include low-grade fever, irritability, drowsiness, and poor appetite. The most accurate assessment of the incidence of these reactions came from a prospective study published by Cody et a161 in 1981, in which adverse reactions were compared in the 48 hours after immunization in children who either received whole-cell DTP or diphtheria and tetanus toxoid vaccine. Local adverse reactions occurred in 64% of DTP recipients. The most common systemic reaction was "fretfulness" (53%) followed by drowsiness (31%) and fever (31%). Among the more troubling of the common adverse reactions associated with DTP is persistent inconsolable crying, observed in 3% of infants in the Cody study.61 In some infants, this crying was described as unusual or "highpitched" in nature. Local reactions were more severe in children who had received prior doses of DTR a trend which has been observed with acellular vaccines as well (as described later). These local and systemic reactions, though common, are generally self-limited in nature and can be generally readily controlled by use of antipyretics. The frequency with which such mild adverse reactions occur can be reduced by premedicating the infant with acetaminophen (15 mg/kg) prior to DTP inoculation.62 More worrisome are the uncommon but severe adverse reactions that occur with whole-cell vaccine. High fever (over 40.5°C) occurs in 1 of 330 vaccine recipients. In some infants, DTP vaccine can precipitate a convulsion, usually in association with fever. Convulsions after DTP immunization are more likely in children with a positive history or family history of convulsions. Although such seizures are distressing to parent and health provider alike, there is no evidence

Curr Probl Pediatr, July 2000

of any association with long-term neurologic sequelae. An enigmatic event occurring after 1 in 1750 DTP doses is the appearance of a hypotonic-hyporesponsive episode, a shock-like state, which usually begins within 12 hours post-inoculation and self-resolves within several hours. The prognosis for complete recovery is excellent, and, fortunately, there does not appear to be any significant long-term sequelae due to hypotonic-hyporesponsive episodes. 63

Contraindications to Whole-Cell DTP Absolute contraindications to whole-cell DTP are anaphylactic reactions associated with previous doses or the development of acute encephalopathy within 7 days after immunization. High fever (> 40.5°C), hypotonic-hyporesponsive episodes, or persistent, inconsolable crying within 48 hours of immunization are considered to be relative contraindications. Seizures within 3 days of vaccination or the presence of a persistent neurologic disorder are considered relative contraindications.64

Can Whole-Cell DTP Lead to Long-Term Neurologic Sequelae? One of the great controversies in pediatric vaccine practice is the question of whether whole-cell DTP vaccine may, in some infants, produce severe, permanent neurologic adverse events. The first published account of acute encephalopathy related to whole-cell pertussis vaccine was in 1933, 53 and multiple anecdotal reports have appeared in the literature in the ensuing decades, ttowever, there have been few controlled studies that have carefully scrutinized this purported link. Since DTP is typically administered in infancy, at a time when many neurologic syndromes and disorders become manifest, establishing a direct, causal link between pertussis vaccination and adverse neurologic sequelae has been difficult. One of the best efforts at defining the risk of serious neurologic sequelae after whole-cell pertussis vaccination came from the National Childhood Encephalopathy study, a casecontrolled study performed in England in the mid 1970s. 65 This study focused on children between the ages of 2 months to 3 years, who were admitted to the hospital with any acute neurologic disease. The study determined which vaccines had been administered in the month prior to the acute neurologic event, and compared cases with age- and sex-matched controls. A total of 11 children had significant neurologic events

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associated with long-term sequelae, leading to the estimation that acute encephalopathy occurs with a frequency of 1 case per 140,000 vaccinations, with a risk of long-term brain damage that occurs at a rate of 1 case per 310,000 doses. The authors concluded that the data suggested, but did not prove, a link between pertussis immunization and permanent neurologic damage. In retrospect, the risk estimates from the National Childhood Encephalopathy study may have been overstated. Of the original 11 cases, there were 4 children with infantile spasm, 2 children with disseminated viral infection, and 1 child with Reye syndrome, diagnoses incompatible with vaccine-associated injuries. Evaluation of the data from this and other studies by the Institute of Medicine led to the conclusion that "there is evidence consistent with causation" for the development of acute encephalopathy without permanent brain damage after inoculation with whole-cell vaccine. 66 However, the Institute of Medicine did not conclude that pertussis vaccine was causally linked to permanent brain damage. The results of the National Childhood Encephalopathy study have been exhaustively scrutinized, and the data subjected to extensive analysis and debate. Most authorities today, including the American Academy of Pediatrics, agree that whole-cell vaccine is not associated with permanent neurologic sequelae. Other associations of concern have included putative links to DTP and infantile spasm, 67 sudden infant death syndrome,68 and abnormal speech and intellectual development.69 However, because these problems present early in life, at an age when infants are receiving DTP vaccine, it is obvious that associations are confounded by the young age of infants at the time of receipt of DTP vaccine. Careful analysis of the published reports linking DTP to these events has failed to establish causality, and the Institute of Medicine panel concluded that no causal relationship existed. Nevertheless, the extensive negative publicity about the risks of whole-cell pertussis, which this and other reports engendered , no doubt contributed to efforts to develop safer pertussis vaccines.

Acellular Pertussis Vaccines

History of Acellular Vaccines Although whole-cell pertussis vaccines were extremely effective in controlling pertussis, concerns about the safety of these vaccines was a major driving force in the

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development of safer alternatives. Concerns about vaccine safety were most acute in Japan, where, as noted previously, pertussis immunization rates fell from 85% to 15% after reports in 1975 of 2 deaths in children who received whole-cell vaccine. 58 This suspension of the pertussis immunization programs led to a major resurgence in disease, and was the driving force behind development of so-called "acellular" pertussis vaccines. Manufacturing techniques were developed that allowed the acellular vaccines components to be purified (by sucrose gradient centrifugation) from B pertussis culture supernatants. 7° Importantly, this modification allowed the majority of the endotoxin to be removed from the vaccine preparation. A further modification was the detoxification of PT, with the use of formalin. 71 These modifications were extremely effective in decreasing the reactogenicity of pertussis vaccines. These early acellular vaccines were of 2 general types: a vaccine that contained approximately equal amounts of PT and FHA, developed by the Research Foundation for Microbial Diseases of Osaka University (designated as "Biken" vaccine), and a family of vaccines which contained more FHA and significantly less PT (developed by Takeda Chemical Institutes, and hence designated as "Takeda" vaccine).72-74 These early acellular pertussis vaccines developed in Japan were first subjected to clinical efficacy evaluation in a trial in Sweden in 1986.75 This study was sponsored by the National Institutes of Health (NIH), and Sweden was selected because of the epidemic nature of disease in what was at that time an unimmunized population. The study was a randomized, placebocontrolled trial comparing a 2-component Biken vaccine (containing PT and FHA) and a single-component PT vaccine. Infants were immunized between 5 and 11 months of age, with a second dose given 8 to 12 weeks after the first dose. Efficacy for both vaccines was less than anticipated, with an overall efficacy of 69% for the 2-component vaccine, and 54% for the single-component vaccine. These results gave the initial impression that acellular vaccines were less efficacious than whole-cell vaccines, and as a result Japan was the only country in which they were licensed. In retrospect, this conclusion was premature and inappropriate. Importantly, no whole-cell vaccine control was included in the experimental design. Had whole-cell vaccine control been included in the study, the acellular vaccines may have compared very favorably, since historical estimates of efficacy of wholecell vaccine studies may in fact overstate the true effi-

Curr Probl Pediatr, July 2000

Licensed Acellular Pertussis-Continuing Vaccines as of January 2000

ACEL-IMUNE

Wyeth-Lederle Laboratories

PT, FHA, FIM2 PRN

All 5 doses

Tripedia

Aventis-Pasteur

PT, FHA

Initial 4 doses*

TriHIBit

Aventis-Pasteur

PT, FHA, ActHIB

4th Dose* ONLY

Infanrix

SmithKline Beecham

PT, FHA, PRN

Initial 4 doses*

PT

Initial 4 doses*

Laboratories

Certiva

North American Vaccine

FIG 4. Currently available acellular pertussis vaccines. Licensed products containing acellular perlussis vaccine indicated for childhood immunization schedule include DTaP products and a single combination DTaP/Hib vaccine.

cacy of whole-cell vaccines. The study design calling for a 2-dose regimen was also, in retrospect, problematic. Had a more standard 3-dose regimen been used, these acellular vaccines would have almost certainly shown better efficacy. However, the most clear-cut evidence of the efficacy of these vaccines came from pertussis surveillance studies after widespread implementation in Japan. By 1989, reported pertussis cases were near an all-time low, providing striking evidence for the effectiveness of these vaccines. 74

Newer Acellular Vaccines The Japanese experience stimulated efforts in other countries to develop acellular pertussis vaccines. Overall, over 20 different acellular vaccines have been developed. Importantly, these vaccines all differ with respect to number and quantity of components, methods of purification, and method of PT inactivation. Fig 4 summarizes the details of the composition of the acellular pertussis vaccines currently licensed for use in the United States. Each acellular vaccine is considered in detail below. All vaccines are combined with diphtheria and tetanus toxoids (hence designated "DTaP" vaccines) and adsorbed onto aluminum salts. Although the list is current at the time of this review, it is likely that additional products will become available in the years ahead. ACEL-IMUNE (Wyeth-Lederle Vaccines). The first acellular vaccine to be licensed in the United States was ACEL-IMUNE, manufactured by Wyeth-Lederle

Curr Probl Pediatr, July 2000

Laboratories (St Davids, Pa). This product was licensed in December 1991, for use as the fourth and fifth (booster) doses in the pertussis immunization series. 76 A reformulated product was licensed for use in the infant primary series in December 1996. 77 ACEL-IMUNE is unique among the currently licensed products in both the number of components it contains (the only acellular vaccine to contain PT, FHA, PRN, and fimbrial agglutinogen 2 (FIM2) antigens) as well as the relative proportions of PT and FHA (it contains significantly less PT and significantly more FHA than do other acellular pertussis vaccines). ACEL-IMUNE combines the acellular pertussis vaccine manufactured by Takeda Chemical Industries (Osaka, Japan) with diphtheria and tetanus toxoids manufactured by Wyeth-Lederle Laboratories. Formaldehyde is used to detoxify the PT component. Tripedia and TriHIBit (Aventis-Pasteur). Tripedia was the second acellular vaccine approved for use in the United Statesl being licensed in August 1992, for the fourth and fifth booster dose in the pertussis immunization series. However, Tripedia was the first acellular pertussis vaccine to receive approval for the infant primary series, in July 1996. 7a This vaccine is manufactured by Aventis-Pasteur (Swiftwater, Pa, formerly Pasteur-Mdrieux-Connaught) and combines the acellular pertussis vaccine manufactured by Biken Corporation (Osaka, Japan) with diphtheria and tetanus toxoids manufactured by Aventis-Pasteur. Tripedia is a 2-component acellular vaccine, which

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contains roughly equivalent amounts of PT and FHA. Formaldehyde is used to detoxify the PT component. The vaccine is currently licensed for only the first 4 doses of the permssis series, but licensure for the fifth dose appears imminent. TriHIBit is the proprietary name of the combination vaccine product obtained when Tripedia is used to reconstitute the Aventis-Pasteur conjugate Hemophilus influenzae type B (Hib) vaccine, ActHIB. Although an attractive option in theory for the infant primary series, there are insufficient data to confirm that these vaccines, when given in combination in a single syringe, allow for a sufficient, protective antibody response to the Hib component. Therefore, use of TriHIBit is restricted to the fourth dose of the pertussis series (typically administered at age 15 to 18 months) and is unfortunately not acceptable for the primary series.

Infanrix (SmithKline Beecham Biologicals). Infanrix was licensed for use in the United States by the FDA in January 1997. 79 This vaccine is a 3-component vaccine containing PT, FHA, and PRN. Infanrix is manufactured by SmithKline Beecham Biologicals (Rixensart. Belgium). Like Tripedia, the vaccine is not licensed for the fifth dose in children who have received four previous doses of DTaE but it appears highly likely that such licensure will be forthcoming before infants currently being immunized as part of the primary series reach the age when the fifth dose is administered. A combination of formaldehyde and glutaraldehyde is used to detoxify the PT moiety. Certiva (North American Vaccine). The most recently licensed acellular pertussis vaccine is Certiva, approved in July 1998. s° The vaccine was developed by the National Institute of Child Health and Human Development (NICHHD) and is manufactured by North American Vaccine (Beltsville, Maryland, formerly known as AMVAX). Several aspects of this product are unique. Certiva consists of a single component, PT, with 40 gg per dose, a higher amount of PT than that found in other acellular pertussis vaccines. Also unique is the manufacturer's method of inactivation of PT, which is detoxified by using hydrogen peroxide. Unlicensed Products in Development. Many acellular pertussis vaccines have been developed and tested in recent years, but for a variety of reasons (including mediocre performance in efficacy trials) have been abandoned. However, some products are likely to go forward for licensure application in the United States in the near future. Acelluvax is a 3-component acellu-

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lar pertussis vaccine containing PT, FHA, and PRN. It is manufactured by Chiron Vaccines (Siena, Italy, formerly Biocine Sclavo). This vaccine is unique insofar as the PT is detoxified by using recombinant molecular technology. The PT gene coding sequence was modified to alter 2 amino acids within the S 1 subunit (Arg 9 ~ Lys and Glu129 --+ Gly), which abolish the enzymatic activity of the S1 subunit while retaining immunogenicity of the PT molecule. 81 PasteurMgrieux-Connaught (Toronto, Ontario, Canada) produces 4- and 5-component acellular pertussis vaccines, combined with diphtheria and tetanus, marketed in Canada and other countries under the proprietary name Tripacel. When combined with conjugate Hib vaccine (ActHIB), the product is marketed under the name Quadracel, and when combined with DT, Hib, and inactivated poliomyelitis vaccine (IPV), the combination product is known as Pentacel. As of April 1998, all Canadian provinces and territories had incorporated Pentacel into their childhood immunization schedule, s2 The recent mandate that only inactivated poliomyelitis vaccine is an acceptable form of immunization against polio in the United States has contributed to the already difficult dilemma of how to administer all of the necessary injections for the wellchild immunization series without turning our children into "pin cushions.''83,84 In addition, the licensure of a conjugate pneumococcal vaccine for the wellbaby vaccine series has further contributed to the dilemma. 85,s6 Therefore, combination vaccines are urgently needed. Once available in the United States, these combination vaccines will be a welcome addition to immunization practice, and will go a long way toward solving the problem of multiple injections.

Safely of Currently Licensed Acellular Pertussis Vaccines The acellular pertussis vaccines uniformly have lower rates of adverse events than do the whole-cell vaccines. In a study comparing adverse event rates among 13 acellular vaccines with 2 whole-cell vaccines, there were significant differences in redness and swelling at the injection site, pain, fussiness, drowsiness, anorexia, and antipyretic use between all of the acellular products and whole-cell vaccines. 87 No acellular vaccine has consistently been found to be more or less reactogenic than the others. Hypotonic-hyporesponsive episodes are observed after acellular vaccine, although with significantly lower frequency than after

Curr Probl Pediatr, July 2000

whole-cell vaccine. A consistent finding with the acellular vaccines is the observation of local redness and swelling with booster doses. Of children given a booster dose (fourth or fifth dose) with acellular vaccine, those primed with acellular vaccine (at 2, 4, and 6 months) have significantly more redness and swelling at the injection site than those primed with whole-cell vaccine. However, the overall reactogenicity is still less than for those who receive a 5-dose series of whole-cell vaccine. Recently, concerns have been raised about the safety of immunizations that contain the preservative, thimerosal. The American Academy of Pediatrics (AAP), with the United States Public Health Service (USPHS), has issued a joint statement alerting clinicians and the public of concern about thimerosal, because of the mercury content of this preservative. 88 Elemental mercury may be neurotoxic, and the developing infant brain may be more susceptible to damage. However, this theoretical risk must be weighed against the very real risk of morbidity and mortality that pertussis carried in infancy. Of the currently licensed acellular pertussis vaccines, all contain thimerosal except for Infanrix. Other manufacturers are currently working on producing thimerosal-free vaccines, which will likely be available within the next year.

Contraindications to Acellular Pertussis Vaccines If whole-cell DTP is contraindicated for any reason in a given patient, then acellular pertussis vaccines are also contraindicated.64 It is not yet clear whether acellular vaccines will reduce the risk of rare, severe neurologic reactions compared with whole-cell vaccines.

Efficacy of Currently Licensed Acellular Pertussis Vaccines As noted above, the first large-scale efficacy trials of acellular pertussis vaccines were conducted in Sweden in 1986. A randomized, double-blind, placebo-controlled trial comparing a Biken 2-component vaccine and a single-component PT vaccine was performed by using a 2-dose regimen, with infants receiving the ftrst dose at 5 to 11 months of age, and a second dose 8 to 12 weeks later. The efficacy of these vaccines appeared disappointing, with a 69% efficacy for the 2component vaccine and only 54% for the single-component vaccine. 75 It was concluded that these acellular

Curr Probl Pediatr, July 2000

vaccines were less effective than whole-cell vaccines in preventing pertussis. In retrospect, however, these conclusions were premature. Only 2 doses of vaccine were given, and it is likely that efficacy would have been much higher if a 3-dose regimen had been used. More significantly, the study lacked a whole-cell control group, making it difficult to draw conclusions about the relative efficacy of the acellular vaccines. All of the acellular pertussis vaccines currently licensed in the United States have been evaluated for efficacy in clinical trials, but it can be extremely difficult to draw comparisons across the various trials. For facilitation of comparison across studies, most efficacy trials use the World Health Organization (WHO) definition of pertussis, which is 21 days of paroxysmal cough, plus laboratory confirmation that the illness is due to pertussis. 89 Even this apparently rigorous definition may be difficult to compare across trials, because of differences in choice and sensitivity of confirmatory assays. In spite of these limitations, some insights into the relative efficacy of acellular vaccines have been gleaned. Of particular importance are the efficacy studies of acellular pertussis vaccines sponsored by the National Institutes of Allergy and Infectious Diseases (NIAID) in Italy and Sweden. 9°,91 These studies were randomized, double-blinded, rigorously controlled trials. Infants were immunized at 2, 4, and 6 months of age, and each study contained both a placebo group and a whole-cell control group. The Italian study compared 2 different 3-component vaccines (containing PT, FHA, and PRN) with whole-cell vaccine, and found an 84% efficacy for both 3-component vaccines, but only a 36% efficacy for wholecell vaccine. The Swedish study revealed an efficacy for a 5-component acellular pertussis vaccine of 85.2%, compared with efficacy for a 2-component vaccine of 58.9% and for a whole-cell vaccine of 48.3%. Several important insights were derived from these trials. One is that the efficacy of traditional whole-cell vaccines was significantly lower than anticipated, and indeed was substantially lower than that of the acellular vaccines. The whole-cell vaccine used in these studies was produced by Connaught Laboratories. Although other studies have suggested that this wholecell vaccine may elicit lower titers to some B pertussis antigens compared with the whole-cell vaccine manufactured by Lederle Laboratories, 92 the low efficacy was nevertheless surprising. The other insight derived from these studies was that acellular vaccines that

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contained 3 or more components, particularly those that contained PRN, appeared to have substantially better efficacy than the 2-component vaccine. Although the results of these studies suggest that the addition of PRN to an acellular vaccine will substantially improve its performance, it must be cautioned that other trials of 2-component and single-component acellular pertussis vaccines have shown efficacy rates similar ,to those reported in the NIAID comparative trials. 93-96However, to date these remain the only controlled studies that have compared multiple acellular vaccines with whole-cell DTR

Conclusion-Which is the Best Acellular Pertussis Vaccine? The Red Book Committee of the American Academy of Pediatrics as well as the American Council on Immunization Practices (ACIP) of the CDC both consider all currently licensed acellular pertussis vaccines to be efficacious and interchangeable. These products are clearly the vaccines of choice today for prevention of pertussis. The ACIP recommends exclusive use of acellular pertussi s vaccines for all doses of the pertussis vaccine series. However, controversy exists regarding which acellular vaccines are most efficacious. A recent expert editorial stated that this "present attitude that all DTaP vaccines are equal is a disservice to children and the immunization effort in this and other countries.''97 Although there are undoubtedly differences in the protective efficacy of the currently available acellular vaccines, this statement does not do justice to the tremendous effort put forth worldwide over the past 3 decades to develop safer pertussis vaccines. Surely any incremental deficits in efficacy of any given product are more than made up for by the substantial gains in physician and parental acceptance of the acellular pertussis vaccines. Much remains to be learned about the components of pertussis vaccines responsible for both reactogenicity as well as protective immunity. Much work also needs to be done in defining which vaccines are safe and immunogenic for adult immunization, since this population must be targeted if the goal of pertussis eradication is ever to be realized. However, all of the currently available acellular pertussis vaccines confer protection against disease with a superb safety profile. In an era where vaccine safety is of paramount importance, these vaccines have represented a major and welcome advance in pediatric immunization practice.

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We acknowledge the assistance of Toni Cunningham in preparation of the manuscript. The Division of Infectious Diseases at Children's Hospital, Cincinnati, Ohio, is supported as a Vaccine Treatment and Evaluation Unit (VTEU) site through the National Institutes of Health (NIH AI-45252).

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