Mannose-binding lectin (MBL)-deficiency is associated with an increased susceptibility to pneumococcal infections and other forms of disease. Pneumococcal vaccination is recommended in MBL-deficient patients with recurrent respiratory tract infections (RRTI). The response to pneumococcal vaccination in MBL-deficient individuals has not yet been studied in detail. An impaired response to pneumococcal polysaccharides in MBL-deficient patients might explain the association between MBL deficiency and pneumococcal infections. This study investigates the antibody response to pneumococcal vaccination in MBL-deficient adult patients with RRTI. Furthermore, we investigated whether there was a difference in clinical presentation between MBL-deficient and -sufficient patients with RRTI. Eighteen MBL-deficient and 63 MBL-sufficient adult patients with RRTI were all vaccinated with the 23-valent pneumococcal polysaccharide vaccine and antibodies to 14 pneumococcal serotypes were measured on a Luminex platform. There were no differences observed in the response to pneumococcal vaccination between MBL-sufficient and -deficient patients. Forty-three MBL-sufficient patients could be classified as responders to pneumococcal vaccination and 20 as low responders, compared to 15 responders and three low responders in the MBL-deficient patients. We found no clear difference in clinical, radiological, lung function and medication parameters between MBL-sufficient and -deficient patients. In conclusion, our study suggests that MBL-deficient adults with RRTI have a response to a pneumococcal capsular polysaccharide vaccine comparable with MBL-sufficient patients. Moreover, we did not find a clear clinical role of MBL deficiency in adults with RRTI. As MBL deficiency is associated with an increased susceptibility to pneumococcal infections, pneumococcal vaccination might be protective in MBL-deficient patients with RRTI.
Primary immunodeficiencies (PID) are an uncommon cause of recurrent respiratory tract infections (RRTI) in adults. However, a major number of adults with manifest PID present with RRTI [1, 2]. Early diagnosis and initiation of treatment are important for the prognosis of patients with primary immunodeficiencies [3, 4], and therefore many efforts have been made to develop a structured guideline for the diagnosis of patients with PID. This has led to a multi-stage diagnostic protocol developed by the European Society for Immunodeficiencies (ESID) . The ESID protocol was first published in 2006 and subsequently updated in 2012 .
The diagnostic work-up for patients with recurrent ear, nose, throat and airway infections includes measuring the serum activity of the alternative and classical complement pathways and the option to determine the activity of mannose-binding lectin (MBL), a protein that plays a major role in the third complement pathway (i.e. lectin pathway).
MBL is a pattern recognition receptor that can bind mannose and N-acetyl-glucosamine oligosaccharides, which are present on a wide variety of microorganisms. MBL facilitates opsonization of these microorganisms and can activate the complement system via the lectin pathway. Human MBL is encoded by the MBL2 gene on chromosome 10, which comprises four exons. Structural polymorphisms in exon 1 of MBL2 influence the functionality of the MBL protein, whereas polymorphisms in the promoter region quantitatively affect serum MBL levels . Genetic variants that cause MBL deficiency are common in the general population. Whether or not MBL deficiency leads to a survival disadvantage remains a matter of debate .
For instance, MBL-deficient children are at an increased risk for invasive pneumococcal disease, and also nasopharyngeal carriage of Streptococcus pneumoniae is twice as high in MBL-deficient children compared to MBL-sufficient children [9, 10]. In adults, MBL deficiency is associated with a more severe course of pneumococcal infection and an increased risk for invasive pneumococcal disease [11, 12]. In two independent cohort studies, no association between MBL deficiency and susceptibility to community-acquired pneumococcal pneumonia was found [13, 14]. Little is known about the involvement of MBL in the initiation and the effector phase of the antibody response to S. pneumoniae. MBL is considered to have only a minor role in the opsonization of S. pneumoniae [15, 16]. Direct in-vitro demonstration of MBL binding to pneumococci is limited (our own unpublished observations) and no differences in phagocytosis of pneumococci were seen between MBL-sufficient and -deficient patients .
It has been suggested that MBL deficiency, especially in the context of a suboptimal function of other components of the immune system, may contribute to an increased susceptibility for infections [18, 19]. From this viewpoint, it is reasonable that international guidelines recommend the administration of pneumococcal vaccines to MBL-deficient patients with recurrent infections . The response to a pneumococcal capsular polysaccharide vaccine in MBL-deficient individuals has not been studied in detail. We hypothesize that the association between MBL deficiency and pneumococcal infections can be explained by an impaired polysaccharide responsiveness in MBL-deficient compared to MBL-sufficient people.
Therefore, the aim of this study was to investigate the ability of MBL-deficient adults with RRTI to respond to pneumococcal vaccination. To that end, we evaluated the response to pneumococcal vaccination in patients with RRTI. Furthermore, we compared the clinical presentation and immunological laboratory parameters between MBL-sufficient and -deficient adults with RRTI who were evaluated according to the ESID protocol.
Materials and methods
All patients were from St Antonius Hospital, Nieuwegein, one of the largest non-academic teaching hospitals in the Netherlands and a referral centre for patients with pulmonary diseases. All patients referred to the out-patient clinic pulmonary diseases for analysis of recurrent respiratory tract infections in the period September 2009–December 2011 were subjected to an immunological work-up based on the ESID protocol for evaluation of recurrent respiratory tract infections. The work-up included administration of a pneumococcal capsular polysaccharide vaccine (PVX) with measurement of immunoglobulin (Ig)G antibody concentrations to 14 pneumococcal serotypes before and after vaccination (as detailed below). Measurement of functional complement activity, including the MBL pathway (MP), was performed in all patients. Genetic analysis was performed if the MP ≤ 10%.
In total, 117 patients were evaluated prospectively according to the ESID protocol (version 2006). Patients who met the criteria for RRTI, defined as three or more infectious episodes of the respiratory tract per year, were included in the present study. Pneumococcal antibody concentration measurement had to be performed 3–6 weeks after vaccination. Other exclusion criteria were previous lung transplantation, previous pneumococcal vaccination or haematological malignancies. Eventually, 81 patients were included in the present study. A consort diagram of the patient selection procedure is presented in Fig. 1. Patient characteristics were collected retrospectively from the patients' medical records.
Classification of clinical characteristics
Infectious episodes were categorized as sinusitis, bronchitis or pneumonia. Sinusitis was defined as symptomatic inflammation of the paranasal sinuses lasting no longer than 6–8 weeks. Bronchitis was defined as having an excessive mucous secretion . A sputum culture was carried out when possible. Pneumonia was defined as a new infiltrate on a chest X-ray in combination with two of six clinical signs of pneumonia (i.e. cough, sputum production, signs of consolidation on auscultation, leucocytosis or leucopenia, temperature < 35°C or > 38°C or > 10% rods in the differential count and C-reactive protein three times above the upper limit of normal). Pulmonary function testing was carried out according to the standards of the European Respiratory Society . Lung function scores were assigned based on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines . Based on forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) measurement, patients are classified as GOLD stage 0 (no obstruction), stage I (mild obstruction), stage II (moderate obstruction), stage III (severe obstruction) or stage IV (very severe obstruction). Computed tomography (CT)-scan characteristics were derived from patients' radiology reports. Medication use and smoking status were extracted from internal patient reports. Patients who were classified as receiving prednisone therapy started this at least 2 weeks before vaccination [24, 25]. A patient was classified as an ex-smoker in case of smoking cessation for at least 6 months prior to vaccination.
Standard laboratory investigations in all subjects included a blood leucocyte count with differentiation, serum alpha-1 anti-trypsin, serum immunoglobulin and serum IgG subclass-level measurement. Sputum was cultured using standard microbiological techniques.
A commercially available enzyme immunoassay was used to determine the functionality of the classical, alternative and lectin complement pathways (Wielisa®; Wieslab, Lund, Sweden) . Any pathway activity ≤ 10% of normal can be an indication of a complement deficiency and needs additional confirmation. MBL2 genotyping was performed if MP ≤ 10% of normal to confirm a MBL deficiency. Combined haplotypes of functional single nucleotide polymorphisms (SNP) in the promoter region (Y/X) and exon 1 of MBL2 (the coding SNPs B, C and D are denoted as ‘0’ and A is wild-type) were determined using a denaturating gradient gel electrophoresis (DGGE) assay, as described previously . MBL genotypes 0/0 and XA/0 were considered to be MBL-deficient.
Patients were vaccinated intramuscularly with one dose of 23-valent pneumococcal polysaccharide vaccine (Pneumovax 23; Merck, Rahway, NJ, USA) containing 25 μg purified type-specific capsular polysaccharides of 23 pneumococcal serotypes (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F: Danish nomenclature). Blood samples were drawn before and 3–6 weeks after vaccination. Serum samples were stored at −80°C until use.
IgG antibodies against 14 pneumococcal polysaccharides were measured on a Luminex platform (Luminex Corporation, Austin, TX, USA), using a quantitative multiplex immunoassay: the XMAP pneumococcal immunity panel . This assay identifies serotype-specific anti-capsular polysaccharide IgG antibodies to the pneumococcal serotypes 1, 3, 4, 6B, 7F, 8, 9N, 9V, 12F, 14, 18C, 19A, 19F and 23F. All antibody concentrations were calibrated on the international Food and Drug Administration (FDA) reference preparation 89-SF. Samples were analysed as singlets; pre- and post-vaccination sera were analysed in pairs.
Patients were classified as responder or low responder based on their post-vaccination pneumococcal polysaccharide antibody response profile. A positive immune response to a given serotype was defined as a post-vaccination antibody concentration ≥ 1·3 μg/ml or as a ≥ 4-fold antibody concentration increase between the pre- and post-vaccination serum samples . A patient was considered to be a responder if at least 70% of the antibody responses to the serotypes tested (i.e. 10 of 14 tested serotypes) were positive .
Groups were compared with Fisher's exact test, McNemar and analysis of covariance (ancova) where appropriate. Statistical analyses were performed using spss. A P-value < 0·05 was considered to represent a statistically significant difference.
The study group comprised 81 patients, in 20 patients of whom functional MBL pathway activity was less than 10%. Subsequent genotyping showed that 18 patients were MBL-deficient (14 XA/0 and 4 0/0 genotypes). No indication was found for a complement factor deficiency in the alternative or classical complement pathway. The patient characteristics of the total study group and the MBL-sufficient and -deficient subgroups are provided in Table 1. Sex, age and smoking status did not differ between both subgroups. The majority of the patients have an underlying disease, mainly chronic obstructive pulmonary disease (COPD) or asthma. A GOLD score of 0 on a lung function test was significantly more prevalent in the MBL-deficient group (P = 0·0463), but pulmonary pathology on CT-scan images did not differ between both subgroups. Medication use, in terms of gammaglobulin or prednisone, was comparable in both subgroups. Twenty-six patients were using prednisone at the time of their vaccination, with a mean dosage of 10·5 mg/day (range 2·5–50, median dosage 6·25).
Table 1. Clinical characteristics of complement mannose-binding lectin (MBL)-sufficient and -deficient patients.
MBL-sufficient patients (%)
MBL-deficient patients (%)
Total patients (%)
P-values were calculated using Fisher's exact test, based upon the number of patients for whom data regarding a specific parameter was available. Percentages are based on the total number of patients per subgroup. Gammaglobulin substitution therapy was initiated after analysis of the response to pneumococcal vaccination. Prednisone use was scored if the therapy was initiated at least 2 weeks before pneumococcal vaccination. COPD, chronic obstruactive pulmonary disease; CT, computerized tomogtaphy; GOLD, Global Initiative for Chronic Obstructive Lung Disease.
Small airway disease
All patients were vaccinated with the 23-valent pneumococcal polysaccharide vaccine and their antibody response was measured 3–6 weeks after vaccination (median 26 days, interquartile range 23–34 days, range 19–43 days). There were four patients who could already be categorized as responders in the prevaccination stage, two of whom were MBL-deficient. Overall, 58 patients were categorized as a responder to PVX and 23 patients as having a low response. Subgroup analysis showed that 15 of the 18 MBL-deficient patients were responders and three were low responders, compared to 43 responders and 20 low responders in the MBL-sufficient patients. The frequency of response to PVX thus did not differ significantly between the MBL-sufficient and -deficient patient groups (P = 0·2515). The MBL genotypes of the three MBL-deficient patients with a low response to pneumococcal vaccination were XA/YB (n = 2) and XA/YC (n = 1). The MBL genotypes of the 15 MBL-deficient patients with a normal response to pneumococcal vaccination were XA/YD (n = 5), XA/YB (n = 6), YD/YB (n = 2), YD/YC (n = 1) and YB/YB (n = 1).
In order to further analyse the response to pneumococcal vaccination, a detailed comparison was made of serum antibodies to 14 pneumococcal serotypes before and after vaccination. There were no significant differences in prevaccination concentrations between both MBL-sufficient and -deficient patient groups. No significant differences were observed between both groups in the geometric mean antibody levels per serotype after vaccination (Fig. 2), except for the mean antibody level for pneumococcal serotype 4, which was just barely significantly higher in the MBL-deficient patients (P = 0·047 when post-vaccination concentrations are corrected for prevaccination concentrations). There was considerable interpatient variation in the number of serotypes against which patient had protective post-vaccination antibody concentrations. Of the 23 low responders, 16 had a response to fewer than 50% of the serotypes that were measured (i.e. seven serotypes).
The above data indicate that in our cohort of patients with RRTI, 28% can be diagnosed with a (mild) specific polysaccharide antibody deficiency. Four of those patients had an underlying immunodeficiency according to the International Union of Immunological Societies (IUIS) criteria : two patients with hypogammaglobulinaemia (patient 1: IgG = 2·67 g/l and IgA = 0·03 g/l; patient 2: IgG = 3·47 g/l and IgA = 0·45 g/l), one patient with an IgA deficiency (IgA = 0·03 g/l) and one patient with an IgG2 deficiency (IgG = 6·80 g/l and IgG2 = 0·5 g/l). All four patients had a low response to pneumococcal vaccination and were MBL-sufficient.
When comparing the patients with an adequate response to pneumococcal vaccination to patients with a low response for the parameters in Table 1, independent of MBL status, several significant differences are observed for lung function parameters, medication use and serum immunoglobulin levels. All four patients with a humoral immunodeficiency were in the low responder group (P = 0·0053). A lung function score of GOLD 0 was more common in the responder group (26 of 55 responders and two of 21 low responders, P = 0·0029), while a GOLD 2 score was more common in the low responder group (four of 55 responders and seven of 21 low responders, P = 0·0079). Prednisone use was more common in the low responder group (13 of 58 responders and 13 of 23 low responders, P = 0·0072).
Significantly fewer patients receiving prednisone treatment responded with protective antibodies concentrations (≥ 1·3 μg/ml) to four pneumococcal serotypes (1, 8, 14, 23F) compared to the patients without prednisone. Furthermore, the mean post-vaccination concentration was significantly lower in the prednisone user group for pneumococcal serotype 8 (P = 0·004, when post-vaccination concentrations are corrected for prevaccination concentrations).
Serum immunoglobulin levels, including IgG subclass levels, were retrieved for all patients for whom they were available, and shown in Table 2. The levels for each patient were evaluated against the reference lower-level values for the respective immunoglobulin (sub)classes  (IgM = 0·40 g/l; IgA = 0·70 g/l; IgG = 7·0 g/l; IgG1 = 4·90 g/l; IgG2 = 1·50 g/l; IgG3 = 0·20 g/l and IgG4 = 0·08 g/l) and the number of patients with reduced serum levels was determined. No significant differences in immunoglobulin and IgG subclass levels were observed between MBL-sufficient and -deficient patient groups except for serum IgG4 levels, which were reduced only in the MBL-sufficient group (P = 0·0336).
Table 2. Immunoglobulin levels compared between complement mannose-binding lectin (MBL)-sufficient and -deficient patients.
MBL-sufficient patients (%)
MBL-deficient patients (%)
Total patients (%)
P-values were calculated using Fisher's exact test, based upon the number of patients for whom data regarding a specific parameter was available. Percentages are based on the total number of patients per subgroup. Only measurements that were carried out before patients received gammaglobulin are included. Ig, immunoglobulin.
n < 0·40 g/l
n < 0·70 g/l
n < 7·0 g/l
n < 4·90 g/l
n < 1·50 g/l
n < 0·20 g/l
n < 0·08 g/l
Immunoglobulin and IgG subclass levels were also compared between responders and low responders to PVX. There were significantly more low responders with serum IgG levels below the lower-level value (7·0 g/l, 12 of 56 responders and 11 of 20 low responders, P = 0·0028). The same applies to the number of patients with serum IgG1 levels < 4·90 g/l (19 of 58 responders and 13 of 22 low responders, P = 0·0421) and to the number of patients with serum IgG2 levels < 1·50 g/l (14 of 58 responders and 14 of 22 low responders, P = 0·0015).
In addition, several differences between prednisone users and non-prednisone users for immunoglobulin and IgG subclass levels were observed. IgG levels below the lower-level value were more frequent in prednisone users (P = 0·0031), as were IgG1 and IgG2 levels below the lower-level values (P = 0·0306 and P = 0·0010, respectively).
When patients who had reduced IgM, IgA, IgG, IgG1 or IgG2 levels, or were low responders, were excluded for a subgroup analysis, a group of 33 patients remained. This was carried out in order to study the role of MBL deficiency in patients where it was the sole immunological defect. Within this group, there were 25 MBL-sufficient patients and eight MBL-deficient patients. The clinical characteristics of these MBL-sufficient and -deficient patient groups did not differ.
This study is the first to suggest that MBL-deficient patients with RRTI do not demonstrate a significantly lower response to a pneumococcal capsular polysaccharide vaccine compared to MBL-sufficient patients. The only difference in response that was observed between both groups was for the response to pneumococcal serotype 4, which was just significantly higher in MBL-deficient patients. This is most probably a chance finding, and does not suggest a lesser response to pneumococcal serotype 4 in MBL-deficient individuals.
Pneumococcal vaccination in MBL-deficient patients with RRTI is justifiable, based on this quantitative assessment of antibody production in response to vaccination. However, a limitation of the present study is that we studied pneumococcal vaccination only in a diagnostic setting. We evaluated the ability to produce specific antibodies, and not the functionality of those antibodies. Little is known about the effect of MBL on pneumococcal opsonophagocytosis, and this needs to be investigated further, as well as the effect of pneumococcal vaccination on the frequency of pneumococcal infections in MBL-deficient patients.
There were no significant differences in medication use and clinical and immunological laboratory characteristics between MBL-sufficient and -deficient patients in our cohort, except that a GOLD 0 score on lung function tests was significantly more prevalent in MBL-deficient patients and that there were no MBL-deficient patients with reduced IgG4 levels. When we investigated the latter further, it appeared to have been a chance finding. When patients with immunological laboratory abnormalities were excluded, there were no significant differences between MBL-sufficient and -deficient patients.
Our results do not support a clear role for MBL deficiency in adult patients with RRTI, as a different pattern does not emerge in the clinical presentation of MBL-deficient compared to MBL-sufficient patients. Furthermore, the prevaccination antibody concentrations did not differ between both groups, indicating that invasive exposition to pneumococci was not higher in MBL-deficient patients. However, our cohort is somewhat small, and it would therefore be inappropriate to make any definitive conclusions.
Moreover, we could not validate the suggestion that MBL deficiency has an effect only in patients with concomitant immune defects, as there were only three MBL-deficient patients with a low response to pneumococcal vaccination. Also, our cohort comprised only four patients with underlying immune defects, apart from selective antibody deficiencies, who were all MBL-sufficient.
While multiplex techniques have made quantification of the serotype specific antibody response to pneumococcal capsular polysaccharide vaccination easier, conversion of individual antibody concentrations into overall responsiveness is still a subject of debate. We have used the threshold of antibody concentrations ≥ 1·3 μg/ml according to international guidelines . However, the value of 1·3 μg/ml in the guidelines is based on quantification by enzyme-linked immunosorbent assay (ELISA), and has not been validated for Luminex xMAP technologies. Luminex xMAP technology appears to estimate higher concentrations of IgGs to pneumococcal capsular polysaccharides compared to ELISA . Because this has not been shown for all pneumococcal serotypes, it was not possible to correct for the use of Luminex xMAP technology in the classification of antibody responses.
The frequency of low responders to pneumococcal vaccination was comparable with the frequency we found in a previous cohort of patients with recurrent respiratory tract infections . We could not find a suitable cohort of patients in the literature measured by a Luminex multiplex assay with which we could compare the antibody response to pneumococcal vaccination in our patients. The group of low responders as a whole showed a lesser response to all individual pneumococcal serotypes studied compared to the group of responders. We observed considerable interserotype variation in post-vaccination antibody concentrations in both groups, which is in accordance with previous studies on the serotype-specific antibody response to pneumococcal vaccination [33, 34].
A comparison between patients with an adequate response to pneumococcal vaccination and patients with a low response (irrespective of their MBL status) revealed significant differences between both groups for lung function, prednisone use and serum immunoglobulin levels. These differences were compatible with the low responder status, and indicate that patients with a low response to pneumococcal vaccination are predisposed to a more severe clinical presentation than patients with an adequate response.
Significant differences between prednisone users and non-prednisone users for several clinical characteristics indicate that patients who require prednisone have a clinical presentation that sets them apart from other patients. Although prednisone has been widely recognized as a cause of reduced immunoglobulin levels [25, 35], a relation between prednisone use and a low response to pneumococcal vaccination has not been established previously [34, 36, 37]. In this study it was found that prednisone use at the time of vaccination was significantly more prevalent in patients with a low overall response to their pneumococcal vaccination. A low response to pneumococcal vaccination may predispose to a clinical presentation that requires prednisone treatment or, alternatively, prednisone use can affect patients' antibody response negatively. The present study is not capable of discriminating between any of these two options, and further investigation will be needed to find definitive answers.
In summary, this study suggests that MBL status does not play a role for IgG responses to pneumococcal capsular polysaccharide vaccination. Moreover, we did not find a clear clinical role of MBL deficiency in adults with RRTI. As MBL deficiency has generally been associated with an increased susceptibility to pneumococcal infections, pneumococcal vaccination might be protective in MBL-deficient patients with RRTI. The data support other data suggesting that human MBL may be largely redundant for protective immunity in humans .
The authors have no conflicts of interest to disclose.
D. K. and H. V. designed the study; D. K., T. H. and H. V. collected the data; T. H., H. V. and P. Z. analysed the data; J. G. arranged financial support and all authors contributed to the interpretation of the results and the writing of the paper.