Measurement and interpretation of pneumococcal IgG levels for clinical management

Authors

  • P. BALMER,

    1. Manchester Public Health Laboratory, Withington Hospital, Manchester,
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      Current addresses: P Balmer, E Stanford, EB Kaczmarski and R Borrow – Manchester Medical Microbiology Partnership, PO Box 209, Clinical Sciences Building, Manchester Royal Infirmary, Manchester, M13 9WZ, UK. A Melegaro and E Miller – Immunization Division, Health Protection Agency, Communicable Disease Surveillance Centre, 61 Colindale Avenue, London NW9 5EQ, UK.

  • J. NORTH,

    1. Department of Immunology, City Hospital NHS Trust, Birmingham,
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  • D. BAXTER,

    1. St Thomas’ Hospital, Shaw Heath, Stockport,
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  • E. STANFORD,

    1. Manchester Public Health Laboratory, Withington Hospital, Manchester,
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      Current addresses: P Balmer, E Stanford, EB Kaczmarski and R Borrow – Manchester Medical Microbiology Partnership, PO Box 209, Clinical Sciences Building, Manchester Royal Infirmary, Manchester, M13 9WZ, UK. A Melegaro and E Miller – Immunization Division, Health Protection Agency, Communicable Disease Surveillance Centre, 61 Colindale Avenue, London NW9 5EQ, UK.

  • A. MELEGARO,

    1. Immunization Division, Public Health Laboratory Service, Communicable Disease Surveillance Centre, London , UK
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      Current addresses: P Balmer, E Stanford, EB Kaczmarski and R Borrow – Manchester Medical Microbiology Partnership, PO Box 209, Clinical Sciences Building, Manchester Royal Infirmary, Manchester, M13 9WZ, UK. A Melegaro and E Miller – Immunization Division, Health Protection Agency, Communicable Disease Surveillance Centre, 61 Colindale Avenue, London NW9 5EQ, UK.

  • E. B. KACZMARSKI,

    1. Manchester Public Health Laboratory, Withington Hospital, Manchester,
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      Current addresses: P Balmer, E Stanford, EB Kaczmarski and R Borrow – Manchester Medical Microbiology Partnership, PO Box 209, Clinical Sciences Building, Manchester Royal Infirmary, Manchester, M13 9WZ, UK. A Melegaro and E Miller – Immunization Division, Health Protection Agency, Communicable Disease Surveillance Centre, 61 Colindale Avenue, London NW9 5EQ, UK.

  • E. MILLER,

    1. Immunization Division, Public Health Laboratory Service, Communicable Disease Surveillance Centre, London , UK
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      Current addresses: P Balmer, E Stanford, EB Kaczmarski and R Borrow – Manchester Medical Microbiology Partnership, PO Box 209, Clinical Sciences Building, Manchester Royal Infirmary, Manchester, M13 9WZ, UK. A Melegaro and E Miller – Immunization Division, Health Protection Agency, Communicable Disease Surveillance Centre, 61 Colindale Avenue, London NW9 5EQ, UK.

  • R. BORROW

    1. Manchester Public Health Laboratory, Withington Hospital, Manchester,
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      Current addresses: P Balmer, E Stanford, EB Kaczmarski and R Borrow – Manchester Medical Microbiology Partnership, PO Box 209, Clinical Sciences Building, Manchester Royal Infirmary, Manchester, M13 9WZ, UK. A Melegaro and E Miller – Immunization Division, Health Protection Agency, Communicable Disease Surveillance Centre, 61 Colindale Avenue, London NW9 5EQ, UK.


Dr Ray Borrow, Manchester Medical Microbiology Partnership, PO Box 209, Clinical Sciences Building, Manchester Royal Infirmary, Manchester, M13 9WZ, UK.  E-mail: ray.borrow@hpa.org.uk,

SUMMARY

The detection of pneumococcal IgG antibodies is helpful for the evaluation of response to pneumococcal vaccination and need for revaccination. Results generated by the clinical assay which is currently used, in which the 23 valent polysaccharide vaccine is the antigen, were compared to those obtained by a capsular polysaccharide serotype-specific assay that measures IgG antibodies to 9 common serotypes causing invasive disease. Discrepancies in 21/47 (45%) of the results were observed in a direct comparison between the two assays. In each case a positive titre was obtained on the clinical assay but IgG levels on the serotype-specific assay were below the putative protective level of 0·2 µg/ml for at least one of the 9 serotypes assayed. The generation of false positives by the current clinical assay is due to its lack of specificity. Antibodies to C-polysaccharide and all of the 23 serotypes included in the pneumococcal polysaccharide vaccine are incorporated into the final titre whereas the serotype-specific assay adsorbs out noncapsular polysaccharide antibodies. The discrepancies between the two assays highlight the importance of standardized assays that measure putative correlates of protection and demonstrate the need to re-evaluate the current clinical assay. A tool that allows the interpretation of the results of the serotype-specific assay is provided and its potential for assessing individual susceptibility levels to vaccine preventable pneumococcal infection is discussed.

INTRODUCTION

Streptococcus pneumoniae infection is a major cause of invasive bacterial disease in children under the age of 5 years and causes significant morbidity in the elderly. The polyvalent polysaccharide vaccine contains the capsular polysaccharides of 23 serotypes that are currently responsible for the vast majority of invasive pneumococcal infection. However, the utility of this vaccine is limited due to the T cell independent nature of the immune response to polysaccharide antigens and its consequent failure to induce long-term protection and lack of efficacy in children under two years of age. As a result it is only licensed for use in individuals over the age of two years and revaccination after intervals of ≥5 years is recommended [1].

A 7-valent pneumococcal polysaccharide-conjugate vaccine (Prevenar™) has recently been licensed which has been shown to be highly immunogenic in young children with an estimated efficacy of 97·3% against vaccine serotypes following 3 doses [2]. The 7 serotypes included in Prevenar™ are among the most prevalent of those causing invasive pneumococcal disease (IPD) in the targeted age group (<5 years of age) and universal immunization of infants has recently been recommended in the US [3]. In the UK, it has been recently recommended that children less than 2 years of age in high risk groups should receive a primary immunization series of Prevenar ™ and a single dose of the 23-valent polysaccharide vaccine at 2 years of age [4]. The at-risk groups include those with anatomical or functional asplenia; chronic renal disease or nephrotic syndrome; immunodeficiency or immunosuppression due to disease or treatment (including HIV infection); chronic heart, lung and liver disease and diabetes mellitus. It is important to determine accurately the response of individuals in these groups, especially those with a suspected or proven immunodeficiency, to pneumococcal vaccination because if protection is not achieved then antibiotic prophylaxis may be required. Furthermore, given the short-term protection afforded by the 23-valent vaccine, the need for re-vaccination needs to be informed by knowledge of antibody levels likely to predict protection.

Evaluation of vaccine responses requires standardized laboratory assays and definition of immunological correlates of protection. Pneumococcal serotype-specific capsular polysaccharide IgG antibodies are thought to be functional and correlate with opsonic assays [5]. The pneumococcal IgG antibody assay used clinically for evaluating vaccine responses and the need for revaccination employs the 23-valent polysaccharide vaccine as an antigen in the ELISA without any adsorption steps to remove nonfunctional antibodies. Thus, the assay measures levels of IgG to all 23 serotypes in the polysaccharide vaccine and additionally to C-polysaccharide (C-PS) that is contained in all pneumococci and hence the vaccine itself. Antibodies to C-PS are not functional [6]. Adsorption of pre or postvaccination sera with C-PS has been shown to reduce mean levels of anti-pneumococcal polysaccharide antibodies by 15–84%[7]. A further adsorption step, with serotype 22F polysaccharide, to eliminate nonfunctional antibodies thought to recognize the linkage region between the C-PS and the serotype-specific PS, has been shown to further improve specificity which, in elderly patients, can reduce mean antibody levels by a further 80%[8].

The aim of this study was to compare the current clinical assay with the serotype-specific assay which has been recently established at Manchester PHL for analysis of the 7 serotypes included in Prevenar™ (4, 6B, 9 V, 14, 18C, 19F and 23F) and two additional serotypes (1 and 5). These 9 serotypes cover 60% of invasive isolates in England and Wales, all ages combined [9]. Pre- or postvaccination sera were analysed using both assays and results were compared (although the new assay is not looking at all the 23 serotypes included in the vaccine). Furthermore, possible clinical interpretations of the results were addressed and the potential impact  of  any  discrepancy  in  the  laboratory  result  on  the  current advice, and therefore on the patient management, was considered.

METHODS

Specimens received from at-risk groups in the Stockport area (n = 47), requiring assessment of immune status to ascertain whether vaccination or revaccination was necessary, were analysed at Manchester PHL and Immunology Department, City Hospital, Birmingham for anti-pneumococcal IgG levels. The median patient age was 51·9 years (range 2·1–82·5 years).

Current pneumococcal IgG ELISA (Birmingham – clinical assay)

Antibodies against the 23 serotypes present in Pneumovax were measured as described by Hazelwood et al. [10]. Briefly, ELISA plates were coated with a 1 : 100 dilution of Pneumovax. Diluted, unadsorbed sera were then added and bound antibody was detected with monoclonal antibodies directed against IgG (all four subclasses, IgG whole), IgG1 or IgG2. The standards, controls and samples were diluted serially from 1 : 10 to 1 : 640. Optical densities obtained were compared with those from a standard pooled serum and a titre calculated. No international standard is available but the standard used is a serum pool that has been calibrated against the current standards used by National External Quality Assessment Scheme UK. An adult IgG whole titre of less than 50 was reported as low [10] and a response to Pneumovax immunization of less than a four-fold increase in titre was interpreted as inadequate. Concurrent measurement of IgG1 and IgG2 was performed as the IgG response to Pneumovax in young children, IgG2 deficiency, asplenic individuals and postbone marrow transplant patients may be predominantly IgG1 which is considered to be less protective than IgG2.

Serotype-specific pneumococcal IgG ELISA (Manchester)

The WHO consensus pneumococcal IgG ELISA (http:// www.vaccine.uab.edu/WHO2.pdf ) used at Manchester PHL is a modification of Koskela [11–14]. Briefly, test sera are preadsorbed with C-PS (5 µg/ml) to remove nonfunctional antibodies. A further adsorption step is also included with serotype 22F polysaccharide (5 µg/ml). Sera are then incubated on serotype-specific capsular polysaccharide coated plates. The assay is developed using a goat anti-human IgG alkaline phosphatase conjugate and p-nitrophenyl phosphate substrate. Standard curves are generated using an international reference serum (89-S, FDA) and concentrations obtained for unknowns are in µg/ml. The standard reference serum was assayed in duplicate using a 2·5 fold serial titration to seven places on every ELISA plate staring at a dilution of 1 : 300 with a final dilution of 1 : 73242. The standard curve was formed by a using a four-parameter logistic-log model. Test sera were assayed in duplicate using a 2·5 fold serial titration to eight places starting at a 1 : 50 dilution, final dilution of 1 : 30 518. Test sera with high values were repeated starting at a dilution of 1 : 500. Data were analysed by the SOFTmax Pro data analysis program (Molecular Devices, Sunnyvale, CA, USA). The lower limit of detection is 0·01 µg/ml with levels below this assigned a value of 0·005 µg/ml for computational purposes. The percentage CVs for assay variability are as follows: serotype 1 (12·7%), 4 (12·7%), 5 (10·1%), 14 (8·7%), 19F (13·8%), 23F (11·0%), 6B (12·4%), 18C (11·9%) and 9 V (13·9%).

In order to show the potential of the serotype-specific assay, an algorithm was designed to convert the antibody concentrations to each serotype to one result describing the overall susceptibility of the individual to vaccine-preventable invasive pneumococcal disease (IPD) with the serotypes assayed. The contribution of each serotype to the number of vaccine-type IPD cases reported for England and Wales in defined age groups was used, together with the serotype-specific result of the new assay to generate an overall percentage of susceptibility against serotypes covered by the 7 and 9 valent pneumococcal conjugate vaccines, respectively. The 9-valent conjugate vaccine covers serotypes that comprise 60% of the invasive infections in England and Wales, all ages combined and assuming no cross protection between serotypes within serogroups [9]. Ideally, when vaccination with the 23 valent polysaccharide vaccine is being considered, the proportion of 23-valent vaccine-preventable invasive infections to which the individual is presumed protected would be calculated although at present only the IgG antibody concentrations to serotypes in the 9 valent conjugate vaccine is being offered as a clinical service.

RESULTS

Direct comparison of the antibody levels measured by the two assays is not possible because the serotype-specific assay produces a concentration in µg/ml for each serotype and the current clinical assay generates an overall titre against the 23 serotypes (and C-PS) in the unconjugated plain polysaccharide vaccine as the end result. The geometrical mean titre of pneumococcal IgG levels was determined from a healthy UK adult population by the current clinical assay [10] with a level of >640 µg/ml being regarded as ‘normal’ and ≥50 µg/ml being regarded as positive. For the serotype-specific assay, a putative protective level of ≥0·2 µg/ml has been proposed [15].

Figure 1 compares the result for each individual on the current clinical assay versus the result obtained for each individual on each of the serotype-specific assays. The graphs show the ‘normal level’ (640 µg/ml) and the ‘positive threshold’ (50 µg/ml) in the current clinical assay and the putative protective levels for each of the serotypes in the 9-valent serotype-specific assay (≥0·2 µg/ml); a more conservative cut-off of ≥1·0 µg/ml is also shown. Analysis of the results obtained with both assays on the same serum showed that 26/47 (55%) matched because the interpretation of the result indicated protection, either overall on the current clinical assay (≥50 µg/ml) or on each of 9 serotype-specific ELISAs (≥0·2 µg/ml) (data not shown). Only one sample gave a result below the threshold of 50 on the current clinical assay and this was matched by the result on the serotype-specific assay with antibodies to 7/9 serotypes below 0·2 µg/ml. However, in 21/47 (45%) of the samples positive titres were obtained on the current clinical assay that were negative (<0·2 µg/ml) for one or more or the serotypes in the serotype-specific assay. This discrepancy was most common for serotypes 1, 4 and 6B whereas few discrepant results were obtained for serotypes 9 V, 14 and 19F. High antibody levels against 1 or 2 individual serotypes were seen in some sera in which low clinical assay titres were found.

Figure 1.

Comparison of the current pneumococcal IgG assay with the serotype-specific IgG assay. The IgG titre obtained on the current assay was plotted against the log µg/ml obtained for individual serotypes on the serotype-specific assay for each sample. The plots include indications of the relevant protective thresholds for both assays: a titre of 50 and 640 for the current assay and 0·2 and 1 µg/ml for the serotype-specific assay.

Currently there are no guidelines for the interpretation of the results generated with the serotype-specific assay. Revaccination could be recommended if any one of the 9 serotypes measured was below the putative protective level. However, the clinical significance of a serotype-specific IgG level <0·2 µg/ml will depend on the prevalence of IPD due to that serotype in the population. Thus, a common serotype with an IgG level below the putative protective threshold (serotype 14) should be given a higher weighting than a more rare serotype (serotype 5), the weighting being directly related to the prevalence. The proportion of vaccine-preventable IPD due to the 9 measured serotypes was therefore considered and an overall level of susceptibility to IPD caused by those 9 serotypes calculated. One theoretical example is produced in Table 1 where an individual's overall susceptibility level is generated by summating the proportion of vaccine–type IPD infections to which the individual is susceptible (58·3% of those caused by the serotypes in a 9 valent vaccine). Since serotype distribution for invasive disease is age-specific the algorithm takes account of the age of the individual being tested in calculating percentage susceptibility. A second theoretical example is shown in Table 1 where a child's susceptibility to IPD caused by the 7 serotypes in PrevenarTM is calculated.

Table 1.  Theoretical example of analysis of pneumococcal serotype- specific data using the 9- and 7-valent algorithm
SerotypeIgG level to individual serotypeContribution to IPD caused by serotypes in the 9-valent or 7-valent vaccine (%)
  1. †9-valent vaccine for Patient Y and 7-valent vaccine for patient X.

Patient Y (Age group: 65–74 years)
 10·37 5·4
 40·0610·1
 50·37 0·0
 140·0632·1
 19F0·23 6·0
 23F0·2112·5
 6B0·2812·5
 18C1·82 5·4
 9 V0·1516·1
Individual susceptibility (%) 58·3
Patient X (Age group: 1–2 years)
 42·44 4·5
 140·1746·6
 19F0·6514·8
 23F4·38 6·8
 6B0·0814·8
 18C5·07 9·1
 9 V0·28 3·4
Individual susceptibility (%) 61·4

The overall percentage susceptibility to IPD caused by the serotypes in the 9 valent vaccine for the 16 samples with at least one serotype-specific IgG result <0·2 µg/ml is shown in Fig. 2, plotted against the result of the current clinical assay. The sample that was <50 on the clinical assay showed an overall susceptibility of 74% on the serotype-specific assay. The remaining 15 samples had overall susceptibility levels ranging from 5% to 58% with 14 having a level of 10% or greater. There was no evidence of a correlation between the overall susceptibility level and the result on the clinical assay.

Figure 2.

Comparison of data generated on the current clinical assay with data generated by the serotype-specific assay and analysed using the 9-valent algorithm for the 23 samples with a least one result <0·2 µg/ml. The plots show the thresholds of 50 and 640 used for current assay.

DISCUSSION

The major concern over the current clinical assay for the detection of pneumococcal IgG levels is the generation of results falsely interpreted as positive due to the lack of specificity in the assay. The adsorption of the test sera with C-PS and serotype 22F capsular polysaccharide is vital for the specificity of the serotype-specific assay, the latter removing nonfunctional antibodies that recognize a common epitope on pneumococci that is not C-PS. The antigen is thought to be carbohydrate by nature because antibodies that recognize this epitope are absent in infants at 7 months of age [8]. It is possible in the current assay that a strong positive titre achieved may be attributable to nonfunctional antibodies or is generated by serotype-specific antibodies to only one or two serotype(s) capsular polysaccharide(s). Hence, a strong positive titre on the current assay must be interpreted with caution. This lack of specificity creates obvious problems for clinicians in determining patient management strategy and is of particular concern for individuals at high risk of pneumococcal infection. The original description of the current clinical assay [10] included adsorption of antibodies reactive to C-PS, however, this is currently not performed routinely. Inclusion of this adsorption step plus removal of antibodies specific for the cross-reactive phosphocholine bonds may improve the specificity of the clinical assay but the current protective thresholds would have to be re-evaluated.

Replacement of the current clinical assay with the serotype-specific assay is an option. However, serological guidelines need to be derived for the interpretation of results generated by the serotype-specific assay. For the purpose of this study, 0·2 µg/ml has been selected as a putative protective level [15]. A figure of 0·15 µg/ml has been previously shown to be the minimum required for a short-term protective response to Haemophilus influenzae type b conjugate vaccination, although for long-term memory a level of 1 µg/ml in 2–4 years olds is thought to be required [16]. In infants receiving the 7-valent conjugate vaccine at 2, 4 and 6 months, more than 97% achieved ≥0·15 µg/ml of anticapsular antibodies for all serotypes within the vaccine [2]. This correlated with the observed protective efficacy of 97·3%. More recently, on new evidence this antibody concentration has been raised to 0·2 µg/ml [15]. However, the protective level may depend upon the age of the individual, medical and vaccination history. Pre-vaccination levels of antibody have been observed to be greater than 0·5 µg/ml in young healthy adults and elderly vaccine recipients [7,17]. Currently, it appears that 0·2 µg/ml is an appropriate protective level for infants receiving a pneumococcal conjugate vaccine but this may not be appropriate for adults. It is also currently unknown if this level applies to different at risk groups such as immunocompromised subjects. Also, protective levels may differ for different pneumococcal serotypes.

Interpretation of the results generated by the serotype-specific assay is limited due to measurement of IgG levels for only nine serotypes. Had additional serotypes (the remaining ones in the 23-valent polysaccharide vaccine) been measured, the result would have been more useful in terms of a 23-valent vaccination policy. Some of the remaining 14 serotypes are common in older age groups (i.e. 8, 19 A, 3) and not considering them could potentially underestimate an individual's susceptibility level to IPD preventable by the 23-valent vaccine.

The designed algorithm represents an attempt to generate manageable data in the form of an overall susceptibility of infection to the pneumococcal serotypes included in the serotype-specific assay. The prevalence of each of the serotypes in defined age groups is combined with the putative protective level of 0·2 µg/ml to generate the percentage susceptibility to infection. If a threshold of ≥10% overall susceptibility on the serotype specific assay is taken as indicating the need for vaccination, then 21 of the patients would have met this threshold, only one of whom would have been regarded as susceptible on the current clinical assay. Different outcomes for the percentage susceptibility can be obtained depending on whether the 7-valent algorithm or the 9-valent algorithm is used. The7-valent algorithm is designed for analysis of pre- and post-PrevenarTM vaccination samples whereas the 9-valent algorithm is more appropriate for samples with an unknown vaccination history or where response to the 23-valent plain polysaccharide vaccine is being measured.

The fact that a number of sera had low clinical test titres but high serotype-specific IgG concentrations against 1 or 2 serotypes is interesting. This highlights that each serotype is not optimally expressed in the mixture of 23 serotypes in the clinical assay. This could have grave consequences if the clinical assay were to be used for measuring protection against the new 7-valent conjugate vaccine with the possible conclusion of nonresponse.

The currently used pneumococcal assay can define patients at risk of pneumococcal disease by identifying patients with low pneumococcal antibody levels either pre or postvaccination. While it can provide an indication of the risk of pneumococcal infection [18], we have shown that this assay will fail to identify a significant group of patients for whom further medical intervention may be appropriate. The pneumococcal serotype-specific IgG assay has the potential to provide important additional information regarding the protective status of an individual and when taken together with age and clinical history, will provide the clinician with the necessary information to aid patient management. The recent licensure of PrevenarTM will increase the number of individuals receiving a pneumococcal vaccine and the detection of pneumococcal IgG levels will become of increased importance especially in those who are susceptible to pneumococcal infection.

ACKNOWLEDGEMENTS

We thank Daniel Sikkema and Keith Friedman (Wyeth Vaccines, Rochester, NY, US) for their invaluable assistance in establishing the pneumococcal serotype-specific assay at Manchester PHL and David McIntosh (Wyeth Vaccines, Taplow, Maidenhead, UK) for critical reading of this manuscript.

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