Serological evidence of pertussis in patients presenting with cough in general practice in Birmingham.

Three hundred and fifty-six patients in a large suburban practice (registered population 10,400), were diagnosed clinically with acute laryngitis/tracheitis or whooping cough (acute spasmodic cough of three weeks duration) between March 1996 and November 1997. Forty out of 145 who provided specimens for serological testing had evidence of recent infection with Bordetella pertussis. During the study a further 18 patients (mostly younger patients who presented early) had a diagnosis of pertussis confirmed by culture. Fifty-eight cases of pertussis in this population and time period was equivalent to an annual incidence of 330 per 100,000, whereas statutory notifications of pertussis in England and Wales suggested an incidence of less than 4 per 100,000 in the same period. Whooping cough remains an important cause of respiratory illness in all age groups. These results are a reminder for general practitioners to be alert to the diagnosis and a prompt to reconsider national vaccination policy.

Effect of schedule on reactogenicity and antibody persistence of acellular and whole-cell pertussis vaccines: value of laboratory tests as predictors of clinical performance.

The performance of four acellular pertussis vaccines containing between two and five pertussis antigens combined with diphtheria and tetanus toxoids was compared with that of British whole-cell diphtheria/tetanus/pertussis (DTP) vaccine both in laboratory assays for potency, toxicity and immunogenicity, and for reactogenicity and immunogenicity in infants. Clinical responses were evaluated in double blind randomized Phase II trials using 3/5/9 month and 2/3/4 month schedules. The acellular DTPs had much lower toxicity than whole-cell DTP in laboratory tests and were significantly less pyrogenic than whole-cell DTP under both schedules. Local reactions were not consistently lower in acellular than whole-cell vaccinees and varied with the source of the diphtheria and tetanus antigens used. Differences in endotoxin level and content of active pertussis toxin (PT) between acellular DTP vaccines were not clinically significant. The reactogenicity advantage of the acellular vaccines was substantially reduced under the 2/3/4 month schedule due to the reduced reactogenicity of the whole-cell DTP vaccine when given at a younger age. There was no relationship between antigen content measured in micrograms per dose and ELISA antibody responses to filamentous haemagglutinin (FHA) and PT in infants, nor was murine immunogenicity predictive of immunogenicity in humans. Antibody response to PT was attenuated in the whole-cell group under the 2/3/4 month schedule but was unaffected in the group receiving acellular vaccines with individually purified components; antibody response to pertactin (69 kDa antigen) was similar in recipients of the whole-cell and component acellular vaccines under the 2/3/4 month schedule. PT antibody persistence until 4-5 years of age was significantly better in recipients of the component acellular than either the whole-cell vaccine or the co-purified acellular vaccine under the 3/5/9 month schedule. However, diphtheria antitoxin levels were reduced in acellular vaccine recipients under both schedules. Despite significantly lower tetanus potencies of the acellular vaccines in laboratory tests, no differences were found in tetanus anti-toxin responses in children.

Multiple metabolic abnormalities in normal glucose tolerant relatives of NIDDM families.

Non-diabetic first degree relatives of non-insulin-dependent diabetic (NIDDM) families are at increased risk of developing diabetes mellitus, and have been studied to identify early metabolic abnormalities. Hormone concentrations measured by specific enzyme immunoassays were assessed in non-diabetic relatives of North European extraction, and control subjects with no family history of diabetes were matched for age, sex and ethnicity. A 75-g oral glucose tolerance test was conducted and those with newly diagnosed NIDDM were excluded. Basal insulin resistance was determined by homeostasis model assessment (HOMA), and hepatic insulin clearance by C-peptide:insulin molar ratio. Relatives (n = 150) were heavier (BMI: p < 0.0001) than the control subjects (n = 152), and had an increased prevalence of impaired glucose tolerance (15 vs 3%, p < 0.01). The relatives had increased fasting proinsulin levels and decreased C-peptide levels following the glucose load, while insulin levels were increased at all time points. To examine whether the differences in hormone levels were secondary to the differences in glucose tolerance and adiposity, we studied 100 normal glucose tolerant relatives and control subjects pair-matched for age, sex, waist-hip ratio and BMI. The differences in proinsulin levels were no longer apparent. However, the relatives remained more insulin resistant, and had decreased C-peptide levels and C-peptide:insulin ratios at all time points. In conclusion, we have identified several metabolic abnormalities in the normal glucose tolerant relatives, and propose that the decreased hepatic insulin clearance helps to maintain normoglycaemia in the face of combined insulin resistance and decreased insulin secretion.

Constitutive expression of major histocompatibility complex class II antigens on monocytes and B cells correlates with disease in simian immunodeficiency virus-infected rhesus macaques.

Constitutive host factors that influence progression to AIDS are understood poorly. In the macaque model for AIDS, 35 animals infected with simian immunodeficiency virus (SIV) were analyzed for major histocompatibility complex class II antigen expression on blood monocytes and B cells by immunostaining and flow cytometry. Expression varied widely between animals but was constant with time. Level of expression and the proportion of monocytes and B cells that expressed class II were not affected by SIV infection. Significantly more animals developed AIDS in the group with low class II expression than in the group with high expression (P < .001). Progression to disease was faster in animals that expressed poorly (P < .01), and opportunistic pathogens were more common (P < .05). Thus, the constitutive level of class II antigen expression may be a useful prognostic indicator for human immunodeficiency virus disease in humans and may be an important factor in the design of vaccine trials.

Antibody responses and symptoms after DTP and either tetanus or diphtheria Haemophilus influenzae type B conjugate vaccines given for primary immunisation by separate or mixed injection.

The safety and immunogenicity of two conjugate Haemophilus influenzae type B (Hib) vaccines administered either mixed with, or in separate limbs to, a whole-cell DTP vaccine, was compared in infants vaccinated at 2, 3 and 4 months of age. Antibody titres to purified polyribosylribitol phosphate, diphtheria, and to pertussis antigens between infants who received the Hib and DPT vaccines in separate limbs or in the same limbs were similar (P > 0.1) while antibody titres to tetanus toxoid were higher in the later group (P < 0.05). This study demonstrated that both Hib vaccines can be mixed with whole-cell DTP vaccine without reducing immunogenicity of either vaccine or increasing the incidence of adverse reactions.

Antibody responses and reactions to the whole cell pertussis component of a combined diphtheria/tetanus/pertussis vaccine given at school entry.

To measure the clinical effect of adding a whole cell pertussis component to diphtheria/tetanus vaccine (DT) given as a pre-school booster, 190 children aged 4-5 years were randomised by a double-blind method to receive either diphtheria/tetanus/pertussis (DTP) or DT vaccine in a 1:1 ratio at selected clinics in England. The geometric mean antibody titres to each of the three pertussis antigens were at least sixfold higher in the DTP than the DT vaccine group and equalled or exceeded those in infants immediately after primary immunisation with DTP vaccine. There were no significant differences between DTP and DT vaccinated children in their diphtheria and tetanus antitoxin levels. The frequency of large local reactions and systemic symptoms such as crying and a disturbed night was 2-3-fold higher in the DTP vaccinees than in the DT vaccinees. Medication was given to 44% of DTP and 23% of DT vaccinees (p = 0.006). Although the change to whole cell DTP vaccine at school entry would result in good pertussis antibody titres, the 2-3-fold increase in reactogenicity that would be caused may be unacceptable at a time when whooping cough is not circulating widely. Evaluation of acellular DTP vaccines given as a pre-school booster in children vaccinated under the accelerated schedule is planned.

Hormonal and metabolic profiles in subjects with obstructive sleep apnea syndrome and the acute effects of nasal continuous positive airway pressure (CPAP) treatment.

Nocturnal secretion of growth hormone is impaired in patients with obstructive sleep apnea (OSA), but the metabolic consequences have not been reported. We measured blood levels of the hormones insulin, C-peptide, growth hormone, cortisol and glucagon together with the intermediary metabolites of carbohydrate (glucose, pyruvate, lactate, alanine) and lipid metabolism [glycerol, nonesterified fatty acids (NEFA), 3-hydroxybutyrate] in six obese nondiabetic men with OSA on two nights. In the first study, the untreated subjects showed frequent apneas and consequent hypoxemia. The hormone and metabolite concentrations were compared with those obtained on the following night when the subjects were treated effectively with nasal continuous positive airway pressure (CPAP). There were no significant differences in the concentrations of insulin, C-peptide, cortisol or glucagon. We confirmed a marked reduction in growth hormone concentrations in OSA, with a significant increase on the CPAP night. The nocturnal profiles of glucose, pyruvate, lactate, alanine and glycerol showed no differences between the two nights, but concentrations of NEFA and 3-hydroxybutyrate, both products of lipolysis, were significantly greater on the treatment night. Because growth hormone has a lipolytic action, the results suggest that suppression of secretion of growth hormone in untreated OSA results in impaired lipolysis, which is rapidly reversed by nasal CPAP.

Adverse events and antibody response to accelerated immunisation in term and preterm infants.

A study was performed to compare adverse events and antibody response in term and preterm children vaccinated with diphtheria, tetanus, and pertussis vaccine at 2, 3, and 4 months of age. A total of 124 children were recruited and grouped according to gestational age: 37 weeks or more (n = 52), 34 to 36 weeks (n = 40), and less than 34 weeks (n = 32). Study nurses followed up children 24 hours after each vaccination to record temperature, redness, and swelling at the injection site and any systemic symptoms. Proportions of children experiencing adverse events did not differ between groups. Blood samples were obtained six weeks after the vaccination course at which time all children had protective levels of diphtheria and tetanus antitoxins. Geometric mean antibody titres (95% confidence interval) to pertussis toxin were 2754 (2042 to 3715), 5495 (4074 to 7413), and 3690 (2951 to 4677), to filamentous haemagglutinin were 541 (282 to 1023), 951 (537 to 1698), and 614 (426 to 1023), and to agglutinogens 2 and 3 were 12,106 (6918 to 21,380), 21,330 (13,183 to 34,674), and 22,387 (15,136 to 33,113) in children born at a gestational age of less than 34 weeks, 34 to 36 weeks, and 37 weeks or more respectively. These findings support the current recommendations that preterm children are vaccinated at chronological age according to the national schedule.

Localization of antigenic domains on the major subunits of Bordetella pertussis serotype 2 and 3 fimbriae.

Antibody-binding domains on the major subunits of Bordetella pertussis serotype 2 (Fim2) and 3 fimbriae (Fim3) have been identified using synthetic peptides which were screened for recognition by anti-protein monoclonal antibodies (mAbs). The presence of non-contiguous fimbrial epitopes was demonstrated by both anti-Fim2 and anti-Fim3 mAbs, several of which recognized at least two peptides that were discontinuous in the amino acid sequence of the corresponding subunits. The specificity of one mAb, 51/24, directed against Fim2, was investigated by replacement-set analysis of a 10-residue peptide, and revealed that antibody binding to the peptide was dependent on the sequence N94PQ96 which is non-conserved in Fim3. Furthermore, proline at residue 95 was found to be essential for mAb 51/24 binding. The specific anti-Fim3 mAb, AG3A, was found to recognize the 10-residue carboxy-terminal peptide from both Fim3 and, unexpectedly, from Fim2. This result suggests that mAb AG3A serospecificity at the protein level is determined by a conformational constraint which prevents mAb AG3A binding to the Fim2 C-terminal domain. Several free peptides containing amino acid residues which comprise part of the Fim2 and Fim3 epitopic domains were prepared as immunogens. One of these peptides was immunogenic in the mouse, indicating the location of a T-helper cell epitope within the peptide sequence, and induced a strong anti-peptide antibody response. The other peptides each required immunization as a conjugate with a carrier protein for anti-peptide antibody stimulation. All four anti-peptide antibody preparations only weakly recognized fimbriae-coated ELISA plates.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the specificity of the vaccine effect elicited by inactivated simian immunodeficiency virus.

Inactivated, partially purified simian immunodeficiency virus (SIVmac) protected macaques from intravenous challenge with homologous and heterologous strains of SIV that had been grown on human cells but no protection against challenge with monkey peripheral blood mononuclear cell-grown SIVmac was afforded. Human immunodeficiency virus type 1 prepared in an analogous way to the SIVmac vaccine on the C8166 human T cell line protected macaques against challenge with human cell-grown SIVmac. These results suggest that protection may be mediated by xenoimmunization with the vaccine cell substrate proteins. All vaccinated macaques had anti-cell antibodies. Major reactivity to MHC class I antigens was found as well as to a 70-kD protein detectable only under nonreducing conditions.

Intrarectal challenge of macaques vaccinated with formalin-inactivated simian immunodeficiency virus.

Macaques can be protected from intravenous infection with simian immunodeficiency virus (SIV) by vaccination with chemically inactivated virus. However, protection against infection via a mucosal surface has not been demonstrated. We vaccinated four rhesus macaques with formalin-inactivated SIV given intramuscularly. These monkeys, which had remained virus free for 10 months after intravenous challenge with SIV, were given a further dose of vaccine and together with four unvaccinated controls were challenged intrarectally with SIV. Subsequently, virus was isolated from all control animals on five successive occasions, but the vaccinated animals remained free of virus. Proviral DNA could not be detected in peripheral blood mononuclear cells from the vaccinated animals. Preliminary data indicate that vaccinated animals make a local antibody response.

Immunogenicity of combined diphtheria, tetanus, and pertussis vaccine given at 2, 3, and 4 months versus 3, 5, and 9 months of age.

In the UK an accelerated schedule for immunisation against diphtheria, tetanus, and pertussis (injections at 2, 3, and 4 months of age) was introduced in 1990 to replace the more widely spaced schedule of 3, 5, and 9 months. There is concern, however, that the new schedule may be less immunogenic and therefore less protective than the old schedule. We have measured serum concentrations of antibodies against diphtheria, pertussis, and tetanus in infants immunised according to the two regimens. Both schedules resulted in protective concentrations of antibody against tetanus and diphtheria and in satisfactory antibody responses to three pertussis antigens (filamentous haemagglutinin, pertussis toxin, fimbriae). However, immunisation by the old schedule led to significantly higher antibody concentrations against both diphtheria and tetanus than did immunisation by the new schedule (p less than 0.01). In infants immunised with the new schedule, postimmunisation antibody concentrations against tetanus toxoid and against two pertussis antigens (pertussis toxin and fimbriae) were significantly lower in infants in whom preimmunisation (maternally derived) antibody concentrations were high (p less than 0.02). The findings suggest that with an accelerated immunisation schedule maternal antibodies can have an inhibitory effect on the responses to immunisation against tetanus and pertussis.

Persistence of antibody after accelerated immunisation with diphtheria/tetanus/pertussis vaccine.

OBJECTIVE: To determine the persistence of antibody to diphtheria, tetanus, and pertussis in children receiving an accelerated schedule of primary immunisation. DESIGN: Controlled study of antibody testing of blood samples from children immunised according to various schedules: three doses of triple vaccine completed at 8-13 calendar months, 6-7 calendar months, before 6 calendar months, or three doses followed by diphtheria/tetanus before age 2. SETTING: Plymouth Health Authority. SUBJECTS: 129 children aged 4 years who had received three doses of diphtheria/tetanus/pertussis vaccine with or without a diphtheria/tetanus booster. MAIN OUTCOME MEASURES: Diphtheria and tetanus antitoxin concentrations and antibody titres to pertussis toxin, filamentous haemagglutinin, and agglutinogens 2 and 3. RESULTS: All children had protective concentrations of antitoxin to diphtheria and tetanus (greater than or equal to 0.01 IU/ml). There was no evidence of a significant difference in diphtheria or tetanus antitoxin concentrations and pertussis antibody titres in children immunised with an accelerated course (third dose of triple vaccine before 6 months) compared with those who received a longer course (third dose at 8-13 months) with no booster (geometric mean antitoxin concentration 0.411 (95% confidence interval 0.273 to 0.618) v 0.426 (0.294 to 0.616) for diphtheria and 0.358 (0.231 to 0.556) v 0.299 (0.197 to 0.453) for tetanus; geometric mean antibody titres 903 (500 to 1631) v 1386 (848 to 2266) for pertussis filamentous haemagglutinin, 179 (130 to 248) v 232 (167 to 322) for pertussis toxin, and 2002 (1276 to 3142) v 3591 (2220 to 5809) for agglutinogens 2 and 3). CONCLUSION: Immunisation with three doses of triple vaccine at monthly intervals completed before 6 months of age probably provides adequate protection against diphtheria, tetanus, and whooping cough which will persist until the age of the preschool booster.

Phase II trial of whole-cell pertussis vaccine vs an acellular vaccine containing agglutinogens.

An acellular pertussis vaccine containing agglutinogens 2 and 3, pertussis toxin, and filamentous haemagglutinin was developed by the Centre for Applied Microbiology and Research in the UK. 188 infants were entered into a randomised blind trial and received either the acellular or a whole-cell vaccine, combined with diphtheria and tetanus toxoids, in a 3, 5, and 8-10 month schedule. Local reactions were similar in the two groups but significantly fewer infants had systemic symptoms after the acellular vaccine. Mean log-antibody titres to the agglutinogen and toxin components were higher with the acellular than with the whole-cell vaccine. Persistence of antibodies one year after the third dose was also better in the acellular group.

Recognition of pertussis toxin by antibodies to synthetic peptides.

Eight synthetic peptides, selected from the amino acid sequence of pertussis toxin (PT) subunits S1, S2, S3 and S4, were assessed for their ability to induce protein-recognizing and neutralizing antibodies. Seven of these peptides, prepared as conjugates of either keyhole limpet haemocyanin or tetanus toxoid, induced significant levels of antibody, all of which reacted with SDS-denatured PT on Western blots. Six of the antibodies bound to PT-coated ELISA plates; this binding was inhibited by homologous peptide antigen. However, none of the antibodies, including those directed against the N-terminus of subunit S1, were able to attenuate in vivo or in vitro toxin-dependent activity. Further investigation revealed that only one antibody, specific for the C-terminus of S1 (peptide Slc, 237-255), could recognize the conformation of native PT in solution. The other five antipeptide antibodies which reacted with PT-coated ELISA plates did not recognize PT when captured onto ELISA plates via either a monoclonal antibody or fetuin, unless the conformation of the toxin had been relaxed by reduction with dithiothreitol. Conversely, the native PT-recognizing response of peptide Slc did not bind the conformationally relaxed PT molecule. From this study, it appears likely that a peptide capable of inducing PT-neutralizing antibody must closely resemble the conformation of the cognate sequence in the native protein.

Serotyping Bordetella pertussis strains.

The serotyping scheme for Bordetella pertussis, developed in the 1950s, depends on the presence or absence of various strains of three major agglutinogens, two of which have been shown to be fimbrial proteins, and several minor agglutinogens, the biochemical nature of which is unknown. This article reviews the reasons for the confusion which has recently arisen over the nomenclature of the serotype antigens and proposes a simplified serotyping scheme based on the fimbrial components.