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Antibody response to pneumolysin and to pneumococcal capsular polysaccharide in healthy individuals and Streptococcus pneumoniae infected patients
Vaccine 22 (2004) 1157–1161
Antibody response to pneumolysin and to pneumococcal capsular polysaccharide in healthy individuals and Streptococcus pneumoniae infected patients Z. Huo a,∗ , O. Spencer a , J. Miles a , J. Johnson b , R. Holliman b , J. Sheldon a , P. Riches a a
Division of Biochemistry & Immunology, Department of Basic Medical Sciences, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK b Division of Medical Microbiology, Department of Basic Medical Sciences, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK Received 8 May 2003; accepted 26 September 2003
Abstract Background: Animal experiments have shown that antibodies against capsular polysaccharide enhance phagocytosis of pneumococcal bacteria and that antibodies against pneumolysin are anti-inflammatory and prevent pneumococcal invasion. It is not known if an antibody response to pneumolysin can be acquired from natural exposure to pneumococcal bacteria or how the concentration of pneumolysin antibody at the mucosal surface compares with that of antibodies against pneumococcal capsular polysaccharide. This study used an equal potency method to measure specific antibody concentrations against pneumolysin and pneumococcal capsular polysaccharides in order to facilitate comparative estimates of concentrations in saliva and serum. The results may provide experimental information as a basis for an improved pneumococcal vaccine strategy. Results: Healthy individuals had higher IgM and IgG antibody concentrations against capsular polysaccharide than against pneumolysin in both saliva and serum, but for IgA the converse was true. Patients with acute pneumococcal infection had significantly lower concentrations of specific IgG antibodies against both antigens than the healthy group. These patients also had significantly higher concentrations of IgM antibody against both antigens than the healthy control group. Discussion: Healthy individuals acquire a comparatively lower concentration of antibody to pneumolysin than to pneumococcal capsular polysaccharides from natural exposure to pneumococcal bacteria. Patients infected by pneumococcal bacteria have lower specific IgG antibody concentrations to both antigens than healthy individuals. These findings support the view that pneumolysin could potentially be used as a vaccine. For enhanced effectiveness, it could be used as a supplement to Pneumovax® II rather than as a replacement. The two acquired antibodies, i.e. to pneumolysin and to capsular polysaccharide, could then play their protective roles at different stages in the course of pneumococcal infection, and together contribute to an effective immune defence against Streptococcus pneumoniae. © 2003 Elsevier Ltd. All rights reserved. Keywords: S. pneumoniae; Pneumococcal capsular polysaccharide; Pneumolysin; Antibody; Vaccination
1. Introduction Streptococcus pneumoniae is an encapsulated Gram positive bacterium which is part of the normal resident oropharyngeal flora and also an opportunistic pathogen primarily concerned with infections of the upper and lower respiratory tract. Most pneumococcal infections do not occur after prolonged carriage but follow the acquisition of a recently acquired serotype [1], which indicates that the incidence of pneumococcal infection is not only determined by the bacterial pathogenesis but also host immunity. ∗
Corresponding author. E-mail address: [email protected] (Z. Huo).
0264-410X/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2003.09.025
The virulence of S. pneumoniae has largely been attributed to its capsular polysaccharide and pneumolysin. Pneumovax® II, a widely used vaccine, consisting of 23 serotypes of the capsular polysaccharide mixture, aims to boost the level of antibodies against capsular polysaccharide in order to enhance phagocytosis of the bacteria. Although this vaccine has shown a protective function for healthy adults in clinical trials [2], a meta-analysis of trials that involved 13 randomised comparisons with over 45,000 subjects concluded that there is no evidence that pneumococcal vaccination for high-risk groups is of any benefit [3]. Our own previous study supports this since it demonstrated that approximately only one-third of high-risk individuals vaccinated using Pneumovax® II could make an antibody
1158
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response to pneumococcus serotype 14 antigen [4]. Vaccines boosting antibody against capsular polysaccharide are the only antigen-specific vaccines currently available. Pneumolysin is a thiol-activated, membrane-damaging, multifunctional toxin produced by S. pneumoniae and has no serotype specificity. At high concentrations, amino acids within pneumolysin promote oligomerization of the toxin in the cell membranes and the generation of lesions in the cell membrane [5]. The bacterium is then able to pass through these lesions and facilitate the development of a pneumococcal infection [6]. At low concentrations, pneumolysin stimulates the production of inflammatory cytokines like TNF␣ and IL-1 by human monocytes [7]. Pneumolysin inhibits the beating of cilia on human respiratory epithelial cells from the upper respiratory tract [8] and from the alveoli [9], decreases the bactericidal activity and migration of neutrophils [10] and activates the complement system directly [11]. Pneumolysin also possesses the ability to inhibit lymphocyte proliferation and antibody synthesis [12]. Animal experiments have shown that immunisation with pneumolysin delayed or prevented the death of mice after challenge with virulent pneumococci [13]. Pneumolysin plays a critical role in sepsis during the first few hours after infection by enabling pneumococci to cause acute sepsis rather than a chronic bacteraemia [14]. It is not known if an antibody response to pneumolysin can be acquired from natural exposure to pneumococcal bacteria or how the concentration of pneumolysin antibody at the mucosal surface compares with that of antibodies against pneumococcal capsular polysaccharide. This study investigated the concentrations of specific antibodies against pneumolysin and pneumococcal capsular polysaccharides by an equal potency method so that comparative concentrations in the saliva and serum of eight healthy individuals and in serum samples from eight patients with pneumococcal infection could be made. The aim of this investigation was to provide experimental information as a basis for an improved pneumococcal vaccine strategy.
Table 1 Pneumococcal serotypes isolated from blood cultures and clinical diagnosis in eight patients
2. Materials and methods
2.3. Measurement of antibody concentration
2.1. Sample collection
The concentrations of IgM, IgG and IgA against pneumolysin and pneumococcal capsular polysaccharides were measured by an enzyme-linked immunoabsorbent assay (ELISA). The method used in this study was described previously [15]. Briefly, the concentration of coating antigen was 10 g/ml for pneumolysin (purchased from the Molecular Microbiology Unit, Women’s and Children’s Hospital, Adelaide, Australia) or 12 g/ml for a mixture of pneumococcal capsular polysaccharide from 23 serotypes (23PPS, Merck Sharp & Dohme Ltd.). All serum and saliva samples were diluted 1/100 or 1/5 with 0.05% PBS–T20, respectively. Non-specific antibody reaction to cell wall polysaccharide was prevented by absorption of all serum and saliva samples, and materials used for controls and
Eight serum samples (three men and five women) from documented cases of pneumococcal disease were collected at the same time as their blood culture samples. Ethical approval was obtained for the study from the Wandsworth Local Research Ethics Committee, South-West London NHS Health Authority. The age groups of these patients were: <20 years (two patients); 20–30 years (one patient); 40–50 years (two patients); >60 years (three patients). These patients were admitted to hospital due to their symptoms of respiratory system infections. Clinical details and isolated serotypes from blood culture are listed in Table 1. Of the six isolated serotypes, all but one were contained in
Patient no.
Serotype
Clinical diagnosis
1 2 3 4
Not determined S4 Not determined S3
5 6 7 8
S18 S19F S6Aa S9V
Pneumococcal pneumonia Right basal pneumonia Sepsis (R) pleural effusion Septic shock secondary to left lobar pneumonia Left sided pneumonia Pneumonia Pneumonia Chest infection
a
Not in Pneumovax® II vaccine.
the Pneumovax® II vaccine. Saliva and serum samples were taken from similar age-matched eight healthy subjects, five men and three women. Their ages were in the groups: <20 years (one subject); 20–30 years (five subjects); 40–50 years (one subject); 50–60 years (one subject). Serum was separated within 2 h of collection and immediately stored in aliquots at −40 ◦ C until assayed. Saliva samples were collected using a small piece of sponge (a part of a Salivette (Stanstedt, Germany)) placed in the side of the mouth for 5 min to absorb saliva. The sponge was then transferred into a Salivette and centrifuged for 10 min at 600 × g. The collected saliva was aliquoted and then frozen at −40 ◦ C until assayed. 2.2. Measurement of the protein concentrations in saliva The protein concentration of saliva can vary considerably. If these differences are large then it may affect the results of antibody measurement. Albumin was measured to correct for any dilution effect within the saliva. Albumin, IgM, IgG and IgA concentrations were measured in the saliva samples of all the healthy subjects by rate nephelometry (Beckman Array). The coefficient of variation (CV) for this technology is <5%.
Z. Huo et al. / Vaccine 22 (2004) 1157–1161
standards. Two serum pools, one from 50 normal blood donors (NHS) and another from 7 subjects with high IgA concentrations (IgAss), were used as a standard serum for the assays. The two standards were assigned values using an equal potency method [15]. The NHS pool contained 5 mg/l IgM and 25 mg/l IgG antibodies against 23PPS, and 3.2 mg/l IgM and 13.5 mg/l IgG antibodies against pneumolysin. The IgAss pool contained 7.7 mg/l IgA antibody against 23PPS and 4.6 mg/ml IgA antibody against pneumolysin. The method of equal potency allowed a direct comparison between individual subjects of their specific antibody concentrations against 23PPS and pneumolysin.
1159
Healthy
100000
Patient
Concentration (mg/l)
Median
10000
1000
100
IgM
2.4. Statistical analysis
IgG
IgA
Fig. 1. The concentrations of antibodies against pneumolysin and their median in 16 serum samples. The median concentration of antibodies against pneumolysin in the patient group () is higher for IgM than in the healthy group (䊊), but lower for IgG and IgA.
Where values fell outside the upper or lower limits of the standard curve a value was assigned to enable statistical analysis. Data were analysed using SPSS software (version 10). The samples were divided into a healthy group and a patient group. Data that appeared statistically significant was compared using the Mann–Whitney test, and considered significant if P-values were less than 0.05.
Healthy
100000
Patient
Concentration (mg/l)
Median
3. Results 3.1. Concentrations of albumin, IgM, IgG and IgA in the healthy subject’s saliva Albumin, IgM, IgG and IgA concentrations were measured in the saliva samples of the eight healthy volunteers. In most subjects, the concentration of IgM and IgG was below the lower limit of detection (0.04 mg/l for IgM and 9.26 mg/l for IgG), as expected (see Table 2). IgA was found to be within the normal reference range (20–300 mg/l) [16]. The IgA corrected for dilution fell into the range (IgA:albumin) of 1.14–3.63.
10000
1000
100
IgM
IgG
IgA
Fig. 2. The concentrations of antibodies against 23PPS and their median in 16 serum samples. The median concentration of antibodies against 23PPS in the patient group () is higher for IgM and IgA than in the healthy group (䊊) (statistical analysis showed there was no significant difference in IgA response to 23PPS between these two groups), but lower for IgG.
bodies to both pneumolysin and to 23PPS. Comparing the concentrations of the two specific antibodies between the healthy and patient groups, the patients group had a significantly lower level of IgG antibodies to pneumolysin and to 23PPS than the healthy group (P = 0.021 and 0.007), but a significantly higher level of IgM antibodies to pneumolysin and to 23PPS (P = 0.038 and 0.015). The groups showed no
3.2. Antibody concentrations to pneumolysin and 23PPS in serum The antibody concentrations to pneumolysin and to 23PPS in the serum are shown in Figs. 1 and 2. There was considerable variation between individuals in all isotypes of anti-
Table 2 Concentration of albumin, IgM, IgG and IgA in the saliva of eight healthy individuals Subject no. 1 Albumin (mg/l) IgM (mg/l) IgG (mg/l) IgA (mg/l) IgA:albumin
16.9 <0.04 <9.26 61.4 3.63
2 7.20 <0.04 <9.26 32.4 4.5
3 14.0 <0.04 <9.26 38.2 2.72
4 57.5 <0.04 11.9 65.3 1.14
5 38.3 <0.04 <9.26 56.6 1.48
6 22.9 <0.04 <9.26 74.4 3.25
7 14.9 <0.04 <9.26 30.8 2.07
8 25.3 <0.04 <9.26 57.8 2.28
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Concentration (mg/l)
1000
100
Pneumolysin PPS 23
10
Median
1 IgM
IgG
IgA
Fig. 3. The median and antibody concentrations to pneumolysin and 23PPS in the saliva of eight healthy individuals. Healthy individuals have higher IgG antibody concentration to 23PPS than pneumolysin.
significant difference in their IgA response to pneumolysin and to 23PPS (P = 0.279 and 0.05). These results suggest that the antibody responses to the two antigens in the majority of patients are primary immune responses. Comparing the antibody responses to the two antigens within each group, both IgM and IgG antibody responses in serum in the healthy group to 23PPS were significantly higher than to pneumolysin (P = 0.038 and 0.021). However, the patients group showed statistically significant higher responses to 23PPS than to pneumolysin for IgM only (P = 0.015) but not for IgG (P = 0.505). In summary, the patient group had low IgG antibody concentrations to both antigens. 3.3. Antibody concentrations to pneumolysin and 23PPS in the saliva of the eight healthy volunteers In the saliva, IgA was the main antibody response to either pneumolysin or 23PPS (Fig. 3). Compared with the concentrations in the serum of the healthy individuals, the IgA antibodies against both pneumolysin and 23PPS in saliva were significantly higher (P = 0.001). The median concentration of IgA to pneumolysin was higher than to 23PPS (Fig. 3), but there was no statistically significant difference between the two IgA antibody concentrations (P = 0.72). 4. Discussion We have demonstrated previously that the ELISA method using a mixture of multiple-antigens, 23PPS, as a coating antigen has its limitations in measuring antibody response against multiple-antigen complex [15]. Unfortunately, the bacteria isolated from two patients in this study had not been further investigated for their serotype. Pneumococcal infection in the remaining six patients was found to have been caused by a different serotype in each case. Of these serotypes, one (serotype 6A) is not contained in the vaccine. In this situation, we used 23PPS as antigen to demonstrate
the antibody response to a capsular polysaccharide antigen following infection caused by unknown and different serotypes, and not for an evaluation of protective immunity against certain serotype pneumococcal infection. Specific antibodies to pneumolysin and 23PPS can be detected in both healthy individuals and patients. Serum samples from patients presenting with pneumococcal infections were taken at the onset of their infection. This period is not long enough in a primary antibody response for switching antibody isotype. These samples therefore indicate the patients’ current immunity levels to pneumococcus. The low concentrations of IgG antibodies to pneumolysin and 23PPS in the patient group suggest that low levels of specific antibodies may be a predisposing factor in infection. The significantly increased specific IgM antibodies in the patient group suggests that the infections could be caused in most cases by serotypes new to these patients, which is consistent with Johnston Jr.’s view [17], i.e. that most pneumococcal infections are caused by bacteria of a serotype new to the patient, not a carried strain. Since the concentrations of antibodies will vary markedly with the mode of collection of the saliva, the ratio of IgA to albumin was used to monitor the collection of the saliva. All values of this ratio fell into an accepted range (1.14–3.63) so that a comparison of specific antibody responses between individuals could be made. The results from this study showed that the IgA response to pneumolysin was higher than to 23PPS in both serum and saliva, and raises the possibility that humans may make a better IgA antibody response to pneumolysin through vaccination than to 23PPS at mucosal surface. Pneumolysin antibody at the mucosal surface could potentially prevent invasion and serious septicaemia. Antibody which produces protection by preventing entry of bacteria could be more effective than an antibody which aids removal by phagocytosis of bacteria from the tissue and circulation. Pneumolysin could potentially be used as a vaccine. However, for enhanced effectiveness it might be used as a supplement to Pneumovax® II rather than a replacement, and administered
Z. Huo et al. / Vaccine 22 (2004) 1157–1161
as a nasopharyngeal spray, to raise local mucosal immunity. The two acquired antibodies, i.e. to pneumolysin and to capsular polysaccharide, could then play their protective roles at different stages in the course of pneumococcal infection, and together contribute to an effective immune defence to S. pneumoniae.
Acknowledgements The authors wish to thank the financial support from the COGENT Trust. References [1] Alonsodevelasco E, Verheul AFE, Verhoef J, Snippe H. Streptococcus pneumoniae: virulence factors, pathogenesis, and vaccines. Microbiol Rev 1995;59(4):591–603. [2] Smit P, Oberholzer D, Hayden-Smith S, Koornhof HJ, Hilleman MR. Protective efficacy of pneumococcal polysaccharide vaccines. J Am Med Assoc 1977;238:2613–6. [3] Moore RA, Wiffen PJ, Lipsky BA. Are the pneumococcal polysaccharide vaccines effective? Meta-analysis of the prospective trials. BMC Fam Pract 2000;1:1 [on-line at http://www.biomedcentral.com/ 1471-2296/1/1]. [4] Huo Z, Miles J, Harris T, Riches P. Effect of Pneumovax® II vaccination in high-risk individuals on specific antibody and opsonic capacity against specific and non-specific antigen. Vaccine 2002;20:3532–4. [5] Boulnois GJ. Pneumococcal proteins and the pathogenesis of disease caused by Streptococcus pneumoniae. J Gen Microbiol 1992;138:249–59.
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[6] Yoneda K, Coonrod JD. Experimental type 25 pneumococcal pneumonia in rats: an electron-microscopic study. Am J Pathol 1980;99(1):231–42. [7] Houldsworth S, Andrew PW, Mitchell TJ. Pneumolysin stimulates production of tumour necrosis factor ␣ and interleukin-1 by human mononuclear phagocytes. Infect Immunol 1994;62:1501–3. [8] Feldman C, Mitchell TJ, Andrew PW, Boulnois GJ, Read RC, Todd HJ, et al. The effect of Streptococcus pneumoniae on human respiratory epithelium in vitro. Microb Pathog 1990;9:275–84. [9] Rubins JB, Duane PG, Clawson D, Charboeau D, Young J, Zegers BJM. Toxicity of pneumolysin to pulmonary alveolar epithelial cells. Infect Immunol 1993;61:1352–8. [10] Paton JC, Ferrante A. Inhibition of human polymorphonuclear leukocyte respiratory burst, bactericidal activity, and migration by pneumolysin. Infect Immunol 1983;41:1212–6. [11] Mitchell TJ, Andrew PW. Biological properties of pneumolysin. Microb Drug Resist 1997;3:19–26. [12] Ferrante A, Rowan-Kelly B, Paton JC. Inhibition of in vitro human lymphocyte response by the pneumococcal toxin pneumolysin. Infect Immunol 1984;46:585–9. [13] Alexander JE, Lock RA, Peeters CC, Poolman JT, Andrew PW, Mitchell TJ, et al. Immunization of mice with pneumolysin toxoid confers a significant degree of protection against at least nine serotypes of Streptococcus pneumoniae. Infect Immun 1994;62(12):5683–8. [14] Benton KA, Everson MP, Briles DE. A pneumolysin-negative mutant of Streptococcus pneumoniae causes chronic bacteraemia rather than acute sepsis in mice. Infect Immun 1995;63(2):448–55. [15] Huo ZM, Riches PG, Miles J, Harris T. The limitation of Pneumovax® as a detection antigen in measurement of serotypespecific antibodies by ELISA. Ann Clin Biochem 2002;39:398–403. [16] Midford-Ward A, Riches PG, Fifield R, Smith AM. SAS protein reference units. Handbook of clinical immunochemistry. 5th ed. Sheffield: Hallmark Printers; 1996. p. 335–6. [17] Johnston Jr RB. Pathogenesis of pneumococcal pneumonia. Rev Infect Dis 1991;13(Suppl 6):S509–17.
Antibody response to pneumolysin and to pneumococcal capsular polysaccharide in healthy individuals and Streptococcus pneumoniae infected patients Z. Huo a,∗ , O. Spencer a , J. Miles a , J. Johnson b , R. Holliman b , J. Sheldon a , P. Riches a a
Division of Biochemistry & Immunology, Department of Basic Medical Sciences, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK b Division of Medical Microbiology, Department of Basic Medical Sciences, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK Received 8 May 2003; accepted 26 September 2003
Abstract Background: Animal experiments have shown that antibodies against capsular polysaccharide enhance phagocytosis of pneumococcal bacteria and that antibodies against pneumolysin are anti-inflammatory and prevent pneumococcal invasion. It is not known if an antibody response to pneumolysin can be acquired from natural exposure to pneumococcal bacteria or how the concentration of pneumolysin antibody at the mucosal surface compares with that of antibodies against pneumococcal capsular polysaccharide. This study used an equal potency method to measure specific antibody concentrations against pneumolysin and pneumococcal capsular polysaccharides in order to facilitate comparative estimates of concentrations in saliva and serum. The results may provide experimental information as a basis for an improved pneumococcal vaccine strategy. Results: Healthy individuals had higher IgM and IgG antibody concentrations against capsular polysaccharide than against pneumolysin in both saliva and serum, but for IgA the converse was true. Patients with acute pneumococcal infection had significantly lower concentrations of specific IgG antibodies against both antigens than the healthy group. These patients also had significantly higher concentrations of IgM antibody against both antigens than the healthy control group. Discussion: Healthy individuals acquire a comparatively lower concentration of antibody to pneumolysin than to pneumococcal capsular polysaccharides from natural exposure to pneumococcal bacteria. Patients infected by pneumococcal bacteria have lower specific IgG antibody concentrations to both antigens than healthy individuals. These findings support the view that pneumolysin could potentially be used as a vaccine. For enhanced effectiveness, it could be used as a supplement to Pneumovax® II rather than as a replacement. The two acquired antibodies, i.e. to pneumolysin and to capsular polysaccharide, could then play their protective roles at different stages in the course of pneumococcal infection, and together contribute to an effective immune defence against Streptococcus pneumoniae. © 2003 Elsevier Ltd. All rights reserved. Keywords: S. pneumoniae; Pneumococcal capsular polysaccharide; Pneumolysin; Antibody; Vaccination
1. Introduction Streptococcus pneumoniae is an encapsulated Gram positive bacterium which is part of the normal resident oropharyngeal flora and also an opportunistic pathogen primarily concerned with infections of the upper and lower respiratory tract. Most pneumococcal infections do not occur after prolonged carriage but follow the acquisition of a recently acquired serotype [1], which indicates that the incidence of pneumococcal infection is not only determined by the bacterial pathogenesis but also host immunity. ∗
Corresponding author. E-mail address: [email protected] (Z. Huo).
0264-410X/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2003.09.025
The virulence of S. pneumoniae has largely been attributed to its capsular polysaccharide and pneumolysin. Pneumovax® II, a widely used vaccine, consisting of 23 serotypes of the capsular polysaccharide mixture, aims to boost the level of antibodies against capsular polysaccharide in order to enhance phagocytosis of the bacteria. Although this vaccine has shown a protective function for healthy adults in clinical trials [2], a meta-analysis of trials that involved 13 randomised comparisons with over 45,000 subjects concluded that there is no evidence that pneumococcal vaccination for high-risk groups is of any benefit [3]. Our own previous study supports this since it demonstrated that approximately only one-third of high-risk individuals vaccinated using Pneumovax® II could make an antibody
1158
Z. Huo et al. / Vaccine 22 (2004) 1157–1161
response to pneumococcus serotype 14 antigen [4]. Vaccines boosting antibody against capsular polysaccharide are the only antigen-specific vaccines currently available. Pneumolysin is a thiol-activated, membrane-damaging, multifunctional toxin produced by S. pneumoniae and has no serotype specificity. At high concentrations, amino acids within pneumolysin promote oligomerization of the toxin in the cell membranes and the generation of lesions in the cell membrane [5]. The bacterium is then able to pass through these lesions and facilitate the development of a pneumococcal infection [6]. At low concentrations, pneumolysin stimulates the production of inflammatory cytokines like TNF␣ and IL-1 by human monocytes [7]. Pneumolysin inhibits the beating of cilia on human respiratory epithelial cells from the upper respiratory tract [8] and from the alveoli [9], decreases the bactericidal activity and migration of neutrophils [10] and activates the complement system directly [11]. Pneumolysin also possesses the ability to inhibit lymphocyte proliferation and antibody synthesis [12]. Animal experiments have shown that immunisation with pneumolysin delayed or prevented the death of mice after challenge with virulent pneumococci [13]. Pneumolysin plays a critical role in sepsis during the first few hours after infection by enabling pneumococci to cause acute sepsis rather than a chronic bacteraemia [14]. It is not known if an antibody response to pneumolysin can be acquired from natural exposure to pneumococcal bacteria or how the concentration of pneumolysin antibody at the mucosal surface compares with that of antibodies against pneumococcal capsular polysaccharide. This study investigated the concentrations of specific antibodies against pneumolysin and pneumococcal capsular polysaccharides by an equal potency method so that comparative concentrations in the saliva and serum of eight healthy individuals and in serum samples from eight patients with pneumococcal infection could be made. The aim of this investigation was to provide experimental information as a basis for an improved pneumococcal vaccine strategy.
Table 1 Pneumococcal serotypes isolated from blood cultures and clinical diagnosis in eight patients
2. Materials and methods
2.3. Measurement of antibody concentration
2.1. Sample collection
The concentrations of IgM, IgG and IgA against pneumolysin and pneumococcal capsular polysaccharides were measured by an enzyme-linked immunoabsorbent assay (ELISA). The method used in this study was described previously [15]. Briefly, the concentration of coating antigen was 10 g/ml for pneumolysin (purchased from the Molecular Microbiology Unit, Women’s and Children’s Hospital, Adelaide, Australia) or 12 g/ml for a mixture of pneumococcal capsular polysaccharide from 23 serotypes (23PPS, Merck Sharp & Dohme Ltd.). All serum and saliva samples were diluted 1/100 or 1/5 with 0.05% PBS–T20, respectively. Non-specific antibody reaction to cell wall polysaccharide was prevented by absorption of all serum and saliva samples, and materials used for controls and
Eight serum samples (three men and five women) from documented cases of pneumococcal disease were collected at the same time as their blood culture samples. Ethical approval was obtained for the study from the Wandsworth Local Research Ethics Committee, South-West London NHS Health Authority. The age groups of these patients were: <20 years (two patients); 20–30 years (one patient); 40–50 years (two patients); >60 years (three patients). These patients were admitted to hospital due to their symptoms of respiratory system infections. Clinical details and isolated serotypes from blood culture are listed in Table 1. Of the six isolated serotypes, all but one were contained in
Patient no.
Serotype
Clinical diagnosis
1 2 3 4
Not determined S4 Not determined S3
5 6 7 8
S18 S19F S6Aa S9V
Pneumococcal pneumonia Right basal pneumonia Sepsis (R) pleural effusion Septic shock secondary to left lobar pneumonia Left sided pneumonia Pneumonia Pneumonia Chest infection
a
Not in Pneumovax® II vaccine.
the Pneumovax® II vaccine. Saliva and serum samples were taken from similar age-matched eight healthy subjects, five men and three women. Their ages were in the groups: <20 years (one subject); 20–30 years (five subjects); 40–50 years (one subject); 50–60 years (one subject). Serum was separated within 2 h of collection and immediately stored in aliquots at −40 ◦ C until assayed. Saliva samples were collected using a small piece of sponge (a part of a Salivette (Stanstedt, Germany)) placed in the side of the mouth for 5 min to absorb saliva. The sponge was then transferred into a Salivette and centrifuged for 10 min at 600 × g. The collected saliva was aliquoted and then frozen at −40 ◦ C until assayed. 2.2. Measurement of the protein concentrations in saliva The protein concentration of saliva can vary considerably. If these differences are large then it may affect the results of antibody measurement. Albumin was measured to correct for any dilution effect within the saliva. Albumin, IgM, IgG and IgA concentrations were measured in the saliva samples of all the healthy subjects by rate nephelometry (Beckman Array). The coefficient of variation (CV) for this technology is <5%.
Z. Huo et al. / Vaccine 22 (2004) 1157–1161
standards. Two serum pools, one from 50 normal blood donors (NHS) and another from 7 subjects with high IgA concentrations (IgAss), were used as a standard serum for the assays. The two standards were assigned values using an equal potency method [15]. The NHS pool contained 5 mg/l IgM and 25 mg/l IgG antibodies against 23PPS, and 3.2 mg/l IgM and 13.5 mg/l IgG antibodies against pneumolysin. The IgAss pool contained 7.7 mg/l IgA antibody against 23PPS and 4.6 mg/ml IgA antibody against pneumolysin. The method of equal potency allowed a direct comparison between individual subjects of their specific antibody concentrations against 23PPS and pneumolysin.
1159
Healthy
100000
Patient
Concentration (mg/l)
Median
10000
1000
100
IgM
2.4. Statistical analysis
IgG
IgA
Fig. 1. The concentrations of antibodies against pneumolysin and their median in 16 serum samples. The median concentration of antibodies against pneumolysin in the patient group () is higher for IgM than in the healthy group (䊊), but lower for IgG and IgA.
Where values fell outside the upper or lower limits of the standard curve a value was assigned to enable statistical analysis. Data were analysed using SPSS software (version 10). The samples were divided into a healthy group and a patient group. Data that appeared statistically significant was compared using the Mann–Whitney test, and considered significant if P-values were less than 0.05.
Healthy
100000
Patient
Concentration (mg/l)
Median
3. Results 3.1. Concentrations of albumin, IgM, IgG and IgA in the healthy subject’s saliva Albumin, IgM, IgG and IgA concentrations were measured in the saliva samples of the eight healthy volunteers. In most subjects, the concentration of IgM and IgG was below the lower limit of detection (0.04 mg/l for IgM and 9.26 mg/l for IgG), as expected (see Table 2). IgA was found to be within the normal reference range (20–300 mg/l) [16]. The IgA corrected for dilution fell into the range (IgA:albumin) of 1.14–3.63.
10000
1000
100
IgM
IgG
IgA
Fig. 2. The concentrations of antibodies against 23PPS and their median in 16 serum samples. The median concentration of antibodies against 23PPS in the patient group () is higher for IgM and IgA than in the healthy group (䊊) (statistical analysis showed there was no significant difference in IgA response to 23PPS between these two groups), but lower for IgG.
bodies to both pneumolysin and to 23PPS. Comparing the concentrations of the two specific antibodies between the healthy and patient groups, the patients group had a significantly lower level of IgG antibodies to pneumolysin and to 23PPS than the healthy group (P = 0.021 and 0.007), but a significantly higher level of IgM antibodies to pneumolysin and to 23PPS (P = 0.038 and 0.015). The groups showed no
3.2. Antibody concentrations to pneumolysin and 23PPS in serum The antibody concentrations to pneumolysin and to 23PPS in the serum are shown in Figs. 1 and 2. There was considerable variation between individuals in all isotypes of anti-
Table 2 Concentration of albumin, IgM, IgG and IgA in the saliva of eight healthy individuals Subject no. 1 Albumin (mg/l) IgM (mg/l) IgG (mg/l) IgA (mg/l) IgA:albumin
16.9 <0.04 <9.26 61.4 3.63
2 7.20 <0.04 <9.26 32.4 4.5
3 14.0 <0.04 <9.26 38.2 2.72
4 57.5 <0.04 11.9 65.3 1.14
5 38.3 <0.04 <9.26 56.6 1.48
6 22.9 <0.04 <9.26 74.4 3.25
7 14.9 <0.04 <9.26 30.8 2.07
8 25.3 <0.04 <9.26 57.8 2.28
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Concentration (mg/l)
1000
100
Pneumolysin PPS 23
10
Median
1 IgM
IgG
IgA
Fig. 3. The median and antibody concentrations to pneumolysin and 23PPS in the saliva of eight healthy individuals. Healthy individuals have higher IgG antibody concentration to 23PPS than pneumolysin.
significant difference in their IgA response to pneumolysin and to 23PPS (P = 0.279 and 0.05). These results suggest that the antibody responses to the two antigens in the majority of patients are primary immune responses. Comparing the antibody responses to the two antigens within each group, both IgM and IgG antibody responses in serum in the healthy group to 23PPS were significantly higher than to pneumolysin (P = 0.038 and 0.021). However, the patients group showed statistically significant higher responses to 23PPS than to pneumolysin for IgM only (P = 0.015) but not for IgG (P = 0.505). In summary, the patient group had low IgG antibody concentrations to both antigens. 3.3. Antibody concentrations to pneumolysin and 23PPS in the saliva of the eight healthy volunteers In the saliva, IgA was the main antibody response to either pneumolysin or 23PPS (Fig. 3). Compared with the concentrations in the serum of the healthy individuals, the IgA antibodies against both pneumolysin and 23PPS in saliva were significantly higher (P = 0.001). The median concentration of IgA to pneumolysin was higher than to 23PPS (Fig. 3), but there was no statistically significant difference between the two IgA antibody concentrations (P = 0.72). 4. Discussion We have demonstrated previously that the ELISA method using a mixture of multiple-antigens, 23PPS, as a coating antigen has its limitations in measuring antibody response against multiple-antigen complex [15]. Unfortunately, the bacteria isolated from two patients in this study had not been further investigated for their serotype. Pneumococcal infection in the remaining six patients was found to have been caused by a different serotype in each case. Of these serotypes, one (serotype 6A) is not contained in the vaccine. In this situation, we used 23PPS as antigen to demonstrate
the antibody response to a capsular polysaccharide antigen following infection caused by unknown and different serotypes, and not for an evaluation of protective immunity against certain serotype pneumococcal infection. Specific antibodies to pneumolysin and 23PPS can be detected in both healthy individuals and patients. Serum samples from patients presenting with pneumococcal infections were taken at the onset of their infection. This period is not long enough in a primary antibody response for switching antibody isotype. These samples therefore indicate the patients’ current immunity levels to pneumococcus. The low concentrations of IgG antibodies to pneumolysin and 23PPS in the patient group suggest that low levels of specific antibodies may be a predisposing factor in infection. The significantly increased specific IgM antibodies in the patient group suggests that the infections could be caused in most cases by serotypes new to these patients, which is consistent with Johnston Jr.’s view [17], i.e. that most pneumococcal infections are caused by bacteria of a serotype new to the patient, not a carried strain. Since the concentrations of antibodies will vary markedly with the mode of collection of the saliva, the ratio of IgA to albumin was used to monitor the collection of the saliva. All values of this ratio fell into an accepted range (1.14–3.63) so that a comparison of specific antibody responses between individuals could be made. The results from this study showed that the IgA response to pneumolysin was higher than to 23PPS in both serum and saliva, and raises the possibility that humans may make a better IgA antibody response to pneumolysin through vaccination than to 23PPS at mucosal surface. Pneumolysin antibody at the mucosal surface could potentially prevent invasion and serious septicaemia. Antibody which produces protection by preventing entry of bacteria could be more effective than an antibody which aids removal by phagocytosis of bacteria from the tissue and circulation. Pneumolysin could potentially be used as a vaccine. However, for enhanced effectiveness it might be used as a supplement to Pneumovax® II rather than a replacement, and administered
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as a nasopharyngeal spray, to raise local mucosal immunity. The two acquired antibodies, i.e. to pneumolysin and to capsular polysaccharide, could then play their protective roles at different stages in the course of pneumococcal infection, and together contribute to an effective immune defence to S. pneumoniae.
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