Age-Related Differences in Patterns of Increased Bordetella pertussis Antibodies

During the period 2008 to 2010, we identified 11,386 serum samples with increased (positive) levels of antibodies recognizing Bordetella pertussis antigens. We sought to characterize the distribution of positive antibody result patterns in relation to patient age. IgG and IgA antibodies recognizing pertussis toxin (PT) and filamentous hemagglutinin (FHA) were quantified using a multianalyte immunodetection assay. Four mutually exclusive positive result patterns were observed: increased FHA antibodies only, increased PT IgA but not IgG, increased PT IgG but not IgA, and increased PT IgG and IgA. In patients <21 years old, the predominant pattern was increased PT IgG but not IgA, whereas in patients ≥21 years old, it was increased FHA antibodies only. The proportion of positive serum samples exhibiting increased PT IgA but not IgG was <20% in all age categories but showed a stepwise rise with age. The proportions of positive serum samples exhibiting increased PT IgG and IgA were similar (26 to 32%) in the age categories spanning 11 to 60 years of age but lower in the <11- and >60-year-old groups. In 3 of 5 age categories, a significant rise in the proportion of positive serum samples exhibiting increased FHA antibodies only occurred in 2010. Patterns of positive B. pertussis antibody results varied with age. The predominance of increased FHA antibodies only in patients >20 years old suggests that many adults thought to have B. pertussis infections actually have other infections that induce FHA-reactive antibodies. Similarly, the 2010 rise in the frequency of increased FHA antibodies only in some age groups suggests an increase in non-B. pertussis infections.     

Neonatal Liver Disease Associated with Placental Transfer of Anti-mitochondrial Antibodies

Background: Anti-mitochondrial antibody is the diagnostic hallmark of primary biliary cirrhosis. Its role in the aetiology of primary biliary cirrhosis is controversial. Methods: Two cases of neonatal hepatitis seropositive for anti-mitochondrial antibody are described. Anti-mitochondrial antibody Ig isotype and epitopic specificity were investigated by immunofluorescence and enzyme immunoassays. Results: In both infants anti-mitochondrial antibody was of the G class, mainly G1 and G3 subclasses, and reacted with two synthetic peptides reproducing major M2 epitopic regions: inner lipoyl domain pyruvate dehydrogenase complex (PDC)-E2_162-176 and PDC-E3 binding protein (PDC-E3BP)_86-100. One infant also reacted with outer lipoyl domain PDC-E2_35-49, and 2-oxoglutarate dehydrogenase complex (OGDC)-E2_99-113. An identical pattern of reactivity was present in their mothers, indicating the maternal origin of the antibodies. Anti-mitochondrial antibody disappeared in the infants with the disappearance of the liver pathology. Conclusions: The simultaneous disappearance of hepatitis and anti-mitochondrial antibody in the infants suggests a possible causal link between the two.     

Antibodies to Bordetella pertussis adenylate cyclase are produced in man during pertussis infection and after vaccination

Bordetella pertussis produces several potential virulence factors. One of these is an adenylate cyclase which penetrates eukaryotic cells, is activated by calmodulin and generates high levels of intracellular cAMP. We have found that pertussis infection in man leads to production of high titres (2000-8000) of anti-B. pertussis adenylate cyclase antibodies. Such antibodies also are produced after pertussis vaccination. They persist into adulthood, cross the placenta and disappear a few months after birth. The anti-adenylate cyclase antibodies found in human serum during pertussis infection do not neutralise the catalytic and penetrative activities of the enzyme.     

Leukocytosis-Promoting Factor of Bordetella pertussis III. Its Identity with Protective Antigen

Leukocytosis-promoting factor (LPF) was purified from the supernatant of a 5-day liquid culture of Bordetella pertussis strains Tohama and no. 134. The preparations appeared to be filamentous molecules (2 by 40 nm) and contained histamine-sensitizing and hemagglutinin activities in addition to leukocytosis-promoting activity. Active protection tests in mice demonstrated strong protective activities of 80 international protective units per mg of protein in LPF preparations of each strain toxoided by mild Formalin treatment. Passive protection tests in mice with anti-LPF sera indicated that anti-LPF is one of the protective antibodies.     

Triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis

Xu Y, Xu Y, Hou Q, Yang R, Zhang S. Triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis. APMIS 2010; 118: 685–91. A triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis was developed. Three targets were used for amplification in a single tube: the insertion sequence IS481 and the pertussis toxin promoter region (ptxP) for B. pertussis, and the insertion sequence IS1001 for B. parapertussis. The performance of this PCR assay was evaluated in parallel in three single‐target real‐time PCR assays using DNA extracted from B. pertussis and B. parapertussis reference strains and nasopharyngeal swabs taken from 105 patients who had been coughing for more than 7 days. The minimum detection limit of the triplex PCR was one to five colony‐forming units (CFU) of B. pertussis and 1 CFU of B. parapertussis per reaction, and the coefficients of both intra‐ and inter‐assay variation were less than 7%. Results were available within 4 h. Of the 105 nasopharyngeal samples, seven were culture positive and 23 were PCR positive for B. pertussis. All culture‐positive samples were also PCR positive. Our single‐tube triplex real‐time PCR assay proved to be sensitive, specific and suitable for simultaneous detection and discrimination of B. pertussis and B. parapertussis.     

Antigenic Analysis of Bordetella pertussis Filamentous Hemagglutinin with Phage Display Libraries and Rabbit Anti-Filamentous Hemagglutinin Polyclonal Antibodies

Although substantial advancements have been made in the development of efficacious acellular vaccines against Bordetella pertussis, continued progress requires better understanding of the antigenic makeup of B. pertussis virulence factors, including filamentous hemagglutinin (FHA). To identify antigenic regions of FHA, phage display libraries constructed by using random fragments of the 10-kbp EcoRI fragment of B. pertussis fhaB were affinity selected with rabbit anti-FHA polyclonal antibodies. Characterization of antibody-reactive clones displaying FHA-derived peptides identified 14 antigenic regions, each containing one or more epitopes. A number of clones mapped within regions containing known or putative FHA adhesin domains and may be relevant for the generation of protective antibodies. The immunogenic potential of the phage-displayed peptides was assessed indirectly by comparing their recognition by antibodies elicited by sodium dodecyl sulfate (SDS)-denatured and native FHA and by measuring the inhibition of this recognition by purified FHA. FHA residues 1929 to 2019 may contain the most dominant linear epitope of FHA. Clones mapping to this region accounted for ca. 20% of clones recovered from the initial library selection and screening procedures. They are strongly recognized by sera against both SDS-denatured and native FHA, and this recognition is readily inhibited by purified FHA. Given also that this region includes a factor X homolog (J. Sandros and E. Tuomanen, Trends Microbiol. 1:192–196, 1993) and that the single FHA epitope (residues 2001 to 2015) was unequivocally defined in a comparable study by E. Leininger et al. (J. Infect. Dis. 175:1423–1431, 1997), peptides derived from residues of 1929 to 2019 of FHA are strong candidates for future protection studies.     

Use of Alum and Inactive Bordetella pertussis for Generation of Antibodies Against Synthetic Peptides in Mice

We have investigated the efficacy of the combined use of Alum and inactive Bordetella pertussis (iBP) adjuvants for eliciting anti-peptide antibodies. ICR mice were immunized four times at 3-week intervals with each of 7 free (i.e., not conjugated to any carrier) synthetic peptides of 15–17 amino acid residues in Alum + iBP, in the commonly used adjuvant protocols (CFA; CFA (initial) followed by IFA), or in CFA + iBP. Serum samples after 3 and 4 injections were tested by RIA. Use of Alum + iBP greatly increased the production of antibodies for most of the peptides. The results have important implications for human vaccine formulation involving peptides.     

Role of antibodies against Bordetella pertussis virulence factors in adherence of Bordetella pertussis and Bordetella parapertussis to human bronchial epithelial cells

Immunization with whole-cell pertussis vaccines (WCV) containing heat-killed Bordetella pertussis cells and with acellular vaccines containing genetically or chemically detoxified pertussis toxin (PT) in combination with filamentous hemagglutinin (FHA), pertactin (Prn), or fimbriae confers protection in humans and animals against B. pertussis infection. In an earlier study we demonstrated that FHA is involved in the adherence of these bacteria to human bronchial epithelial cells. In the present study we investigated whether mouse antibodies directed against B. pertussis FHA, PTg, Prn, and fimbriae, or against two other surface molecules, lipopolysaccharide (LPS) and the 40-kDa outer membrane porin protein (OMP), that are not involved in bacterial adherence, were able to block adherence of B. pertussis and B. parapertussis to human bronchial epithelial cells. All antibodies studied inhibited the adherence of B. pertussis to these epithelial cells and were equally effective in this respect. Only antibodies against LPS and 40-kDa OMP affected the adherence of B. parapertussis to epithelial cells. We conclude that antibodies which recognize surface structures on B. pertussis or on B. parapertussis can inhibit adherence of the bacteria to bronchial epithelial cells, irrespective whether these structures play a role in adherence of the bacteria to these cells.     

Antibodies to Bordetella pertussis in human colostrum and their protective activity against aerosol infection of mice.

Colostrum samples from Indonesian mothers were assayed for antibodies which agglutinate Bordetella pertussis and for antibodies to the filamentous hemagglutinin and the lymphocytosis-promoting factor of B. pertussis. Agglutinins were assayed by a microtiter method, and 36 of 58 samples tested (62%) had titers above 1:10 (range, less than 1:10 to 1:160). An enzyme-linked immunosorbent assay detected anti-filamentous hemagglutinin in 39 of 60 samples (65%) and anti-lymphocytosis-promoting factor in 26 of 60 samples assayed (43%). A total of 52 samples (87%) were positive for at least one of these antibodies. Pooled colostrum samples were separated by affinity chromatography into fractions enriched secretory immunoglobulin A (sIgA) or IgG and examined for their ability to passively protect suckling mice from aerosol challenge with B. pertussis. Samples (160 micrograms of protein) were given intraperitoneally 90 min before challenge. Death, rate of gain in body weight, and leukocytosis were used as indicators of illness. Colostrum containing anti-lymphocytosis-promoting factor or agglutinins was protective, whereas colostrum lacking these but containing anti-filamentous hemagglutinin gave little protection. The sIgA-enriched and IgG-enriched fractions appeared to be equal in their ability to protect against respiratory challenge with B. pertussis.     

Placental Thrombosis and Fetal Loss After Passive Transfer of Mouse Lupus Monoclonal or Human Polyclonal Anti-Cardiolipin Antibodies in Pregnant Naive BALB/c Mice

In the present study we evaluated the effect of passive transfer of a mouse monoclonal (CAM) or a human polyclonal anti-cardiolipin IgG on pregnancy outcome in BALB/c mice. The mice were immunized through the tail vein immediately after mating with 10 μg of monoclonal or polyclonal anti-cardiolipin antibodies. Two other groups of mice were given a mouse irrelevant monoclonal antibody or normal human polyclonal IgG respectively, at the same dose. In mice immunized with monoclonal or polyclonal anti-cardiolipin antibody we observed a significant increase in the number of fetal resorptions and a significant reduction of the mean weights of the embryos and the placentas. In mice immunized with CAM we also found a significant decrease in the number of healthy pups, while mice infused with human aCL antibody expressed a significant reduction in the fecundity rate. The histological examination showed widespread thrombosis and necrosis in the placentas derived from the mice immunized with the anti-cardiolipin antibodies. The model supports a possible direct pathogenetic effect of anti-phospholipid antibodies in recurrent fetal loss and points out that thrombotic events at placental level can be instrumental in the pathogenesis of the obstetric complications.     

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

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

Polymorphism of repeated regions of pertactin in Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica

Pertactin is an outer membrane protein expressed by Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica that induces protective immunity to Bordetella infections. The immunodominant and immunoprotective epitopes of pertactin include two repeated regions, I and II. Comparison of these two repeated regions showed that B. parapertussis pertactin is invariant, whereas B. pertussis pertactin varies mostly in region I and B. bronchiseptica pertactin varies in both repeated regions I and II, but mostly in region II. These differences may result from specific characteristics of these Bordetella species.     

Bordetella pertussis Infection: Pathogenesis, Diagnosis, Management, and the Role of Protective Immunity

 Whooping cough is presently one of the ten most common causes of death from infectious disease worldwide. Despite a high vaccine uptake, resurgences of this disease have been observed in several countries. Virulence factors of Bordetella pertussis include agglutinogens, fimbriae, P.69/pertactin, pertussis toxin, filamentous haemagglutinin, adenylate cyclase, tracheal cytotoxin, dermonecrotic toxin, lipopolysaccharide, tracheal colonisation factor, serum resistance factor, and type III secretion. Virulence factor expression is regulated by the bvgAS locus, a two-component signal transduction system. The pathophysiologic sequence consists of attachment (fimbriae, P.69/pertactin, tracheal colonisation factor, pertussis toxin, filamentous haemagglutinin), evasion of host defence (adenylate cyclase, pertussis toxin, serum resistance factor), local effects (tracheal cytotoxin), and systemic effects (pertussis toxin). Bordetella pertussis is transmitted by respiratory droplets and causes disease only in humans. Various diagnostic methods are available, including culture, serological methods, and the polymerase chain reaction. Serotyping of isolates to detect agglutinogens 2 and 3 is useful because serotype 1,2 may be associated with higher mortality, and antibodies to these antigens (agglutinins) may be protective in both animals and humans. Immunisation using whole-cell vaccine is effective but is reactogenic. Acellular vaccines containing one to five components are being used increasingly in various countries. Protective immunity to pertussis correlates with high levels of antibody to each of pertactin, fimbriae, and pertussis toxin; however, doubt remains as to the relationship between agglutinogen 3 and fimbria 3, making results of trials investigating these virulence factors difficult to interpret.     

Passive Transfer of Tumour‐Derived MDSCs Inhibits Asthma‐Related Airway Inflammation

Myeloid‐derived suppressor cells (MDSCs), a heterogeneous population including myeloid progenitor and immature myeloid cells, are known to inhibit T cell responses. The issue of whether tumour‐derived MDSCs regulate the immune response in an asthma environment is currently unclear. Here, we have reported that tumour‐derived MDSCs shift the balance back to normal in a Th2‐dominant asthmatic environment. In an ovalbumin (OVA)‐induced mouse asthma model, injected tumour‐derived MDSCs were recruited to the lungs of asthmatic mice by CC chemokine ligand 2 (CCL2). MDSCs transferred into asthmatic mice via i.v. injection suppressed the infiltration of inflammatory cells into the lung, the Th2 cytokine, IL‐4, concentration in bronchial lavage fluid and the serum level of OVA‐specific IgE. Increased TGF‐β1 production in the lung was detected after transfer of MDSCs. The inhibitory effects of MDSCs were reversed upon treatment with an anti‐TGF‐β1 antibody, suggesting dependence of these activities on TGF‐β1. Our findings imply that tumour‐derived MDSCs inhibit the Th2 cell‐mediated response against allergen in a TGF‐β1‐dependent manner. Based on the collective results, we propose that asthma may be effectively targeted using a novel MDSC‐based cell therapy approach.     

Development and Analytical Validation of an Immunoassay for Quantifying Serum Anti-Pertussis Toxin Antibodies Resulting from Bordetella pertussis Infection

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.Despite effective immunization with pertussis vaccines, pertussis remains endemic. Recently reported disease has increased in adolescents and adults, among whom the diagnosis of pertussis is especially problematic (7, 8, 10, 16, 30). Routine surveillance, outbreak investigations, and implementation of control measures have been limited by a lack of validated and standardized tests to identify suspected cases of pertussis (4, 29, 32, 36, 38). By the time health care is sought and pertussis is suspected, isolation of Bordetella pertussis by culture or confirmation by PCR is uncommon (24, 29, 31, 36). Although detecting the physical presence of the bacterium is difficult, antibody titers directed to B. pertussis tend to be elevated during the later phase of illness and persist after the infection is resolved, so the development and validation of a simple and rapid serodiagnostic test has practical utility for diagnosing pertussis infections. The ability to confirm outbreaks quickly using a serologic test has the potential to conserve resources that have been used for outbreaks mistakenly attributed to pertussis (4). Additionally, the routine availability of a serodiagnostic test for pertussis would provide significant public health benefit by furnishing public health officials with a more accurate assessment of the burden of disease in the United States and with a better understanding of the role of adolescents and adults in the transmission of pertussis. Acellular pertussis vaccines for adults and adolescents have recently been licensed (5, 6); however, the absence of readily available, validated and standardized tests to confirm suspected cases in these older age groups has hampered the collection of the epidemiological data required to guide developers and public health officials in effective utilization of these vaccines (11, 12, 32). A serodiagnostic test could supply these data and allow the design and evaluation of control strategies.A large body of evidence is now available to demonstrate that measurement of specific antibodies could assist in the laboratory confirmation of pertussis (8, 13-15, 17, 20); however, the criteria defining the infection threshold are not well agreed upon by international and national health organizations. One proposal for threshold values was based on the measurement of antibodies against pertussis toxin (PT), filamentous hemagglutinin, and fimbria types 2 and 3 in a population of more than 6,000 U.S. residents of ages 6 to 49 years who participated in the Third National Health and Nutrition Examination Survey (2). Based on the mixture modeling of these data to identify hypothesized exposure groups, an anti-PT immunoglobulin G (IgG) level of >94 ELISA units (EU)/ml was proposed as the diagnostic cutoff point for recent infection, with a lower value of >49 EU/ml as an intermediate cutoff that suggested possible infection (3). Alternate diagnostic thresholds have been developed and applied. Specifically, the Massachusetts State laboratory has utilized a cutoff value of 200 EU/ml for almost 20 years (23), and De Melker et al. (9) adopted a value of 125 EU/ml for routine use in The Netherlands. Thus, the above studies established a variety of threshold cutoffs for anti-PT titers that range from 49 to 200 EU/ml. Final assessment of these proposed diagnostic cutoff points requires a prospective clinical study including patients with confirmed B. pertussis infection. By establishing accurate cutoff values for anti-PT titers for patients currently or recently ill, serological detection may provide a qualitative assessment of whether a test sample has anti-PT titers that are higher or lower than appropriately defined positive and negative control values.Despite these potential benefits, no Food and Drug Administration (FDA)-approved diagnostic assays are currently available for the serodiagnosis of B. pertussis infection, and none of the published methods (1, 9, 17, 19, 23, 25-27, 33-35, 37) have been demonstrated to be readily transferable to public health laboratories. Thus, the overall goal of this project is to develop a simple and readily transferable enzyme-linked immunosorbent assay (ELISA) for the measurement of anti-PT IgG in human serum samples that subsequently could be subjected to an appropriate clinical assessment. A single-serum dilution-based ELISA procedure with ready-to-use reagents was designed and optimized to quantify the anti-PT range thought relevant for diagnosing late-stage pertussis infections. We describe the initial assay development, initial evaluation of the prototype kit by an interlaboratory collaborative study, and assay validation study.     

Expression of pertussis toxin in Bordetella bronchiseptica and Bordetella parapertussis carrying recombinant plasmids.

Pertussis toxin is produced only by strains of Bordetella pertussis. Cloned genes encoding pertussis toxin from B. pertussis were transferred into Bordetella bronchiseptica and Bordetella parapertussis by conjugation. These transconjugants expressed pertussis toxin at levels comparable to those expressed by B. pertussis. The toxin made by these strains was biologically active in the Chinese hamster cell clumping assay, contained all five subunits, and was mostly periplasmic. Toxin expression appeared to be modulated in the same way as are the vir-regulated genes of B. pertussis. Introduction of these plasmids into B. pertussis failed to produce hypertoxigenic strains. Instead, these transconjugants underwent plasmid loss, gene deletions, or conversion to the avirulent phase.     

Kinetics and sensitivity of ELISA IgG pertussis antitoxin after infection and vaccination with Bordetella pertussis in young children

Sera from 96 young children in a vaccine trial were analysed for kinetics of ELISA IgG anti‐pertussis toxin (anti‐PT) after a laboratory‐verified pertussis infection. The antibody decay curves after infection were biphasic and similar in shape to those after vaccination. The change from a rapid to a slower decay after the peak occurred about 4–5 months from the first day of cough. In a group of children given a two‐ or a five‐component acellular pertussis vaccine the proportion of sera above the tentative cut‐off values for anti‐PT of 20, 50 or 100 EU/ml 12 months after onset of the infection were 19%, 0% and 0% respectively. Corresponding figures for a whole‐cell or placebo vaccine group of infected children were significantly higher, 73%, 39% and 30%, i.e. the antibody decay after infection in young children depends on vaccination status as well as on the pertussis vaccine given. In a large group of non‐infected children vaccinated with the same five‐component acellular vaccine 13%, 0% and 0% had sera above 20, 50 and 100 EU/ml at 12 months after the third vaccine dose and all were below the minimum level of detection 2 years after vaccination. In conclusion, knowledge about anti‐PT kinetics is essential for the interpretation of seroepidemiological data but hardly offers the possibility to establish valid cut‐off values for anti‐PT in single sample serology. An option would be to identify a grey zone between the positive and negative ends of the distribution for follow‐up testing by a second serum.     

Specific Secondary Biological Properties of Purified Rabbit Immunoglobulin M and Immunoglobulin G Antibodies to Brucella abortus and Bordetella pertussis

Rabbit immunoglobulin (Ig)M and IgG antibodies to Brucella abortus and Bordetella pertussis were isolated as purified products and their specific secondary biological activities were compared. IgM antibodies were found to be more active than IgG proteins in inducing agglutination and sensitization of B. abortus for the complement-dependent bactericidal effect and in inhibiting B. pertussis-induced lymphocytosis in the mouse. IgM and IgG antibodies were found to be equally effective in inducing agglutination of B. pertussis suspended in a colloidal solution. These data parallel previous work to indicate that IgM antibodies to bacterial surface antigens are more efficient than IgG molecules in initiating biological processes concerned with the inactivation of these pathogens.