Research Article

Immunology and Cell Biology (2000) 78, 254–258; doi:10.1046/j.1440-1711.2000.00910.x

Pertussis antibody levels in infants immunized with an acellular pertussis component vaccine, measured using whole-cell pertussis ELISA

Mg Hanlon1, R Nambiar1, Am Kakakios1, P McIntyre2, Mv Land3 and Pl Devine3

  1. 1Department of Immunology and Infectious Diseases, New Children's Hospital, New South Wales, Australia
  2. 2National Centre for Immunization Research, Westmead, New South Wales, Australia
  3. 3PanBio Australia, Windsor, Queensland, Australia

Correspondence: Mark Hanlon BSc MASM, BSc MASM, Senior Hospital Scientist, Department of Immunology and Infectious Diseases, PO Box 3515, Parramatta, NSW 2124, Australia. Email: markh@nch.edu.au

Received 13 October 1999; Accepted 8 February 2000.

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Abstract

A commercially available whole-cell pertussis IgG ELISA was used to test the response of 137 2-month-old infants to immunization with a trivalent acellular pertussis vaccine. The pre-immunization geometric mean (GM) IgG index was 6.96 (95% confidence interval (CI) 5.88–8.04) and the postimmunization GM index was 13.16 (95% CI 12.20–14.11), P < 0.001. Eighty percent of subjects (110/137) had a significant 1.5-fold increase of pertussis IgG index (97/137, 71%) or a postimmunization IgG index > 10 (93/137, 68%). In single antigen ELISA, 83% showed at least a fourfold increase in pertussis toxin-specific IgG (PT-IgG) and 91% showed an increase in IgG specific for filamentous haemagglutinin (FHA-IgG). Four percent had high pre- immunization antibody levels (index > 20), likely to reflect recent maternal exposure to pertussis. This correlated with a smaller increase in pertussis IgG index. A decline in pertussis IgG index postimmunization occurred in 17/24 infants (71%) whose pre-immunization IgG index was > 10. This postimmunization pertussis IgG index was not significantly different to that of infants with a low pre-immunization index. A similar trend was noted with PT-IgG and FHA-IgG results. The whole-cell ELISA can detect a response to acellular pertussis vaccination in most infants if both antibody index and degree of seroconversion are calculated and at least one criterion is satisfied.

Keywords:

acellular vaccine, enzyme-linked immunosorbent assay, immunoglobulin G, pertussis

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Introduction

In the last decade, a number of acellular pertussis vaccines have been shown to be immunogenic in infants.1, 2, 3, 4, 5, 6 All of these studies have used ELISA serological assays with the individual purified pertussis components as antigens. Such assays are not available to routine diagnostic laboratories, which use commercial pertussis antibody ELISA with a whole-cell extract of pertussis as the antigen or, less often, locally prepared assays using whole-cell pertussis extract. The antigenic composition of the extract in these assays is undefined.

In the present study, antibody response using a commercially available whole-cell pertussis IgG antibody ELISA kit was compared with specific ELISA to pertussis antigens contained in the vaccine, in infants receiving their primary course of immunization with a three-component acellular pertussis vaccine.

The aims of the study were to test whether a whole-cell assay could detect a significant rise in IgG antibodies in infants following immunization with an acellular pertussis component vaccine and to evaluate the influence of maternal antibodies on postimmunization antibody levels.

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Materials and Methods

One hundred and eighty healthy infants were recruited at birth and given Infanrix® (SmithKline Beecham Biologicals, Rixensart, Belgium) at 2, 4 and 6 months of age. The vaccine contains inactivated tetanus and diphtheria toxoids and three acellular pertussis antigens: pertussis toxin, filamentous haemagglutinin and pertactin.

Blood samples were collected prior to immunization at 2 months of age and again at 7 months of age. Samples were tested for IgG antibodies to tetanus toxoid, diphtheria toxoid, Haemophilus influenzae type b capsular antigen (Hib), whole-cell pertussis IgG, antipertussis toxin (PT) and antifilamentous haemagglutinin (FHA) IgG. In 43 infants there were insufficient pre- or postimmunization samples for testing, leaving 137 infants for evaluation. Analysis of available pre- or postimmunization samples from these 43 excluded subjects for tetanus, diphtheria, Hib, PT, FHA and pertussis antibody titres (data not shown) suggested that their antibody responses were not different to those of the 137 subjects included and were therefore unlikely to affect the results.

Blood samples were allowed to clot at room temperature for 1 h and then were centrifuged at 1000 g for 10 min to recover the serum. Serum was frozen until pre- and postimmunization samples could be tested in parallel using a commercially available kit ELISA (PanBio Pertussis IgG ELISA, Cat BPG-100; Windsor, QLD, Australia). The serology was performed according to the manufacturer's instructions. Serum was diluted 1/100 in the diluent provided, transferred to the Bordetella pertussis antigen-coated microwells and incubated for 20 min at 37°C (100 muL/well). After washing with phosphate-buffered saline containing 0.05% Tween 20, bound IgG was detected via a 20 min, 37°C incubation with antihuman IgG peroxidase (100 muL/well). The plate was then washed and a 10 min incubation with tetramethylbenzidine substrate (100 muL/well) performed. The reaction was stopped by the addition of 100 muL of 1 mol/L phosphoric acid to each well and the strips were read using a microtitre plate reader at a wavelength of 450 nm. Results were calculated according to the manufacturer's instructions. An IgG reference serum that has a specified pertussis IgG index of 10 (cut-off calibrator) is provided by the manufacturer, and the ELISA index is defined as (10 times sample absorbance)/calibrator absorbance. An index less than 10 is considered not suggestive of recent pertussis infection and an index greater than 10 or a 1.5-fold increase in the index between acute and convalescent samples is consistent with recent exposure to pertussis.

The 137 paired samples were also tested in ELISA assays that used PT or FHA as the antigen. Briefly, PT (1 mug/mL) or FHA (0.5 mug/mL) were coated on microtitre plates overnight at 4°C. The plates were washed five times with PBS-Tween 20 (PBST) and then incubated with 1% bovine serum albumin in PBS (PBST-BSA) as a blocking agent. Serum samples were diluted 1:50 in PBST-BSA for the anti-PT IgG assay and 1:100 for the anti-FHA IgG assay. Diluted samples were incubated at 37°C for 60 min, washed five times in PBST and then incubated with horseradish peroxidase-conjugated antihuman IgG diluted 1:1000 in PBST-BSA for 60 min at 37°C. After washing five times in PBST, ortho-Phenylene diamine (o-PD) substrate was added and the plates were incubated at 37°C for 30 min before being read at 405 nm. A strongly positive sample collected from an adult who had recently recovered from pertussis infection was serially diluted for eight dilutions, to provide a standard curve. An international reference serum (JNIH-10) obtained from the National Institute for Biological Standards and Control (NIBSC, Herts, UK) was used as the reference and sample titres were calculated from the standard curve using the parallel line reference method of Reizenstein et al.7 Four secondary (internal) controls were included in each batch. Interassay coefficients of variation (CV) were less than or equal to 15%.

Statistics for examining significance were calculated using SPSS® (SPSS Inc., Chicago, IL, USA), after consulting a biostatistician. The Wilcoxon signed rank test, Mann–Whitney U-test, or Chi-squared test were used, as appropriate. This study was approved by the Ethics Committee of the New Children's Hospital, Westmead, NSW, Australia.

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Results

Whole-cell pertussis IgG

There was a significant rise in antibody to whole cell pertussis between pre- and postimmunization samples using a number of different measures. The geometric mean (GM) of the pertussis IgG index in pre-immunization samples was 6.96 (95% confidence interval (CI) 5.88–8.04), increasing 1 month after the third immunization to 13.16 (95% CI 12.20–14.11; P < 0.001 using the Wilcoxon signed rank test). The median increase of IgG index was 2.7-fold (range 0.8–17.2). In more than 62% of infants, the increase was greater than twofold. Only 2% of infants whose pre-immunization IgG index was < 10 failed to show a rise in antibody level.

Among the cohort of 137 infants, 97 (71%) achieved a 50% increase in pertussis IgG index and 93 (68%) had a postimmunization IgG index greater than 10. Using these criteria in combination, 110 (80%) showed an immunization response. Thus, the kit detected a significant increase in antibody in the majority of study subjects. Twenty-four of the 137 (18%) pre-immunization samples collected at 2 months of age had an index of 10 or greater, of which six (4.4%) had an index of 20 or over. This suggests that almost 20% of the mothers had a level of IgG to whole cell pertussis antigen thought to be consistent with recent exposure.

There was a strong negative association between high levels of whole-cell pertussis IgG pre-immunization antibodies and the fold increase in postimmunization levels measured in this assay. Figure 1, a Bland–Altman plot, shows an inverse relationship between the pre-immunization index and the increase in pertussis IgG index postimmunization.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Bland–Altman plot of postimmunization pertussis IgG index minus pre-immunization index versus pre-immunization pertussis IgG index measured using the commercial whole-cell ELISA. This figure shows the magnitude of the change in pertussis IgG index compared with the pre-immunization pertussis IgG index. The y-axis is the increase (or decrease, if negative) in IgG index after immunization and the x-axis is the pre- immunization IgG index, plotted on a log scale. The slope of the regression line is – 8.38, indicating an association of high pre-immunization IgG index with lower postimmunization IgG indices (P < 0.001).

Full figure and legend (11K)

In the 113 infants whose pre-immunization IgG index was < 10, the median pre-immunization pertussis IgG index was 4.22 (range 0.82–17.28), the median rise in IgG index was 2.7-fold and the median postimmunization IgG index was 12.31 (range 5.09–38.57). Only three of 113 infants (2.6%) had a lower postimmunization index.

In the 24 infants whose pre-immunization IgG index was greater than or equal to 10, only seven (29.2%) showed an increase in pertussis IgG index postimmunization. This was significantly different to 97.3% (110/113) who showed an increase among those infants with lower pre-immunization titres (chi2 = 73.89, P < 0.0005). In the six infants with pre-immunization IgG index greater than or equal to 20, none had an increase in pertussis IgG index. These six had an average twofold decline in IgG index. Of importance, however, the GM of the postimmunization pertussis IgG index was 13.6 (95% CI 8.8 –18.3), which was not significantly different to the infants with low pre-immunization IgG index (Figure 2).

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

This boxplot illustrates the change in pertussis IgG index in relation to pre-immunization pertussis IgG index. The geometric mean (GM) of the pertussis IgG index of group 1 (pre-immunization IgG index < 10) increased, while that of group 2 (pre-immunization IgG index > 10) decreased. However, there is no significant difference (P = 0.73) between the post-immunization GM pertussis IgG index of these two groups. Boxes are interquartile ranges. Bars show the range from 10th to 90th percentiles. (circle), Outliers; (——), mean; (——), median.

Full figure and legend (15K)

Pertussis subcomponent assays

Most infants had strong antibody responses to pertussis antigens, with 83% of postimmunization PT-IgG titres and 91% of the postimmunization FHA-IgG titres increasing more than fourfold. In contrast to the whole cell pertussis assay, only two infants (1.5%) had high pre-immunization PT-IgG titres, and two different subjects (1.5%) had high pre-immunization FHA-IgG titres. These results suggest that at least four (3%) of the mothers had recent exposure to pertussis.

Three subjects showed no change or a decline in anti-PT IgG after immunization and two showed a decline in anti-FHA-IgG. Only one infant had high pre-immunization titres in all three assays, and this subject had a decline in pertussis IgG index and anti-FHA-IgG, but a small increase in anti-PT IgG after immunization. There was a slight but highly significant association between high pre-immunization anti-PT IgG or anti-FHA IgG and lower post immunization titres (P < 0.006, Mann–Whitney U-test).

Comparison of whole-cell pertussis IgG with pertussis component assays

The difference in antibody response between those with low and high pre-immunization whole-cell pertussis antibody is summarized in Table 1. The infants whose pre-immunization whole-cell IgG index was high tended to have higher pre-immunization PT-IgG (P = 0.005) and FHA-IgG titres (P = 0.03), and smaller increases in PT-IgG (P = 0.006) and FHA-IgG (P = 0.045) after immunization. The difference in fold increase in IgG index between the infants with low pre-immunization IgG index and those with high pre- immunization IgG index was highly significant (P < 0.001).


There was no significant difference between those with high and those with low pre-immunization IgG indices in terms of the geometric mean pertussis IgG index (P = 0.42), the anti-PT IgG (P = 0.77) or the anti-FHA IgG (P = 0.39) after immunization (Table 1). The pre-immunization PT-IgG and FHA-IgG were significantly different from the postimmunization PT-IgG and FHA-IgG.

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Discussion

While there have been many published studies of acellular pertussis vaccine trials in which immunogenicity is assessed using single-antigen ELISA, we know of none in which the serology was performed using a whole-cell pertussis ELISA. In those studies in which the effects of maternal antibodies on immunization response and postimmunization antibody levels were examined, single-antigen ELISA were used to measure the responses.

The antigenic material used in the whole-cell pertussis ELISA assay is a complex mixture of pertussis antigens that vary in immunogenicity. In contrast, the antigens used in the anti-PT and anti-FHA ELISA are pure preparations of single antigens. This may account for the different results we found between the whole-cell ELISA and the single-antigen ELISA assays performed on pre- and postimmunization sera of infants vaccinated with an acellular pertussis component vaccine.

Our results showed that the whole-cell ELISA detected more subjects (18%) with a high or moderately high level of pertussis-specific IgG in pre-immunization samples than did the anti-PT IgG ELISA (2%) or the anti-FHA IgG ELISA (2%). Although this difference is quite large, it could be the result of the sensitivity of the whole-cell ELISA for antibodies to other pertussis antigens, such as the lipo-oligosaccharide (LOS), which is very immunogenic, or adenylate cyclase, pertactin, fimbrial antigens or any of a combination of other pertussis antigens.

The high pertussis IgG in pre-immunization samples is possibly due to placental transfer of maternal antibodies. High levels of pertussis antibodies in the mothers could occur as a result of previous vaccination of the mothers, but the findings of Cattaneo et al.8 suggest that pertussis antibodies will be at their lowest in this age group. Consequently, it is more likely that these titres are the result of infection by or exposure to Bordetella pertussis and this is consistent with reports that whooping cough was epidemic in Australia for several years prior to the present study.9

Few studies have been published on the use of whole-cell ELISA to measure pertussis antibodies. Mertsola and colleagues10, 11, 12, 13 have assessed the performance of whole-cell ELISA in a number of pertussis outbreaks and have compared that assay with single-antigen assays and with ELISA assays that use a pool of purified antigens. Their conclusion was that the antipertussis IgG measured in a whole-cell ELISA is quite likely to be high in the 'acute' phase blood sample of vaccinated individuals, and that IgG seroconversions are therefore not commonly detected, even if there is a rise in pertussis IgG antibodies.

Our data show that only 71% of infants produced a significant response (greater than or equal to 1.5-fold increase in IgG index) to immunization with an acellular pertussis vaccine measured by a whole-cell IgG ELISA kit, compared with 83% showing a fourfold or greater rise in anti-PT IgG and 91% showing a fourfold or greater rise in anti-FHA IgG. In comparison with the single-antigen ELISA, the whole-cell ELISA underestimates the proportion of subjects who respond to the acellular pertussis vaccine when the criteria for a response is an increase in antibody levels.

Like Mertsola et al.,10, 11, 12, 13 we found a high level of pre-existing placentally acquired antipertussis IgG, which was not unexpected during a period in which pertussis was epidemic. This affects the magnitude of the vaccine response, as measured in the whole-cell ELISA assay if only the fold-increase in IgG index is considered. However, the number of responders was increased (80%) by including subjects satisfying the manufacturer's cut-off IgG index of greater than or equal to 10. The results of the whole-cell ELISA assay indicated a strong negative influence of maternal antibody on the magnitude of the immune response. However, this is likely to be an artefact, because the postimmunization IgG indices of subjects with a high pre-immunization index were similar to those of subjects with a low pre-immunization IgG index. Our results reflect the fact that the whole-cell ELISA detects pertussis antibodies that are specific for antigens other than PT or FHA, although a significant component of the antibody response measured by the whole-cell ELISA may be the FHA-IgG antibodies. The contribution of the PT-IgG antibodies to the whole-cell IgG index is lower. A larger study will be needed to confirm this.

The single-antigen ELISA results show a small, but highly significant (P < 0.01) negative association with vaccination response. Other studies have used single antigen ELISA only, and either have14 or have not15, 16 shown a significant influence of pre-immunization antibody. One study17 examined the effect of high cord blood antibody titres on antibody response to a whole cell pertussis vaccine and found a negative influence of maternal antibodies on postimmunization PT IgG, but no significant influence on postimmunization anti-FHA IgG levels.

Assuming that high pre-immunization IgG indices are due to placental transfer of maternal antibodies and that antibodies produced by immunization are likely to be very low in women of child-bearing age, high titres are likely to be due to maternal exposure to B. pertussis. Thus, it is possible that the whole-cell ELISA is more sensitive than single-antigen ELISA to pertussis-specific antibodies produced after infection. The sensitivity of the whole-cell ELISA to antibodies specific for pertussis antigens other than PT and FHA, as seen in the pre-immunization samples, is desirable in an assay intended for the diagnosis of infection.

Pertussis IgG measurements are often used for diagnosis of pertussis infection in the very young infants in preference to pertussis IgA, owing to the poor IgA response of infants and young children to B. pertussis. Our results show that the kit is capable of detecting maternally acquired pertussis IgG, so care is required in the interpretation of pertussis IgG results for the diagnosis of pertussis infection in the very young.

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Acknowledgements

We thank Dr J Peat, Dr Janaki Amin and Ms Margie Gruca for their assistance with the statistical analyses and Dr Melanie Wong and Dr Jill Forrest for their editorial suggestions.