Vaccination against encapsulated bacteria in hereditary C2 deficiency results in antibody response and opsonization due to antibody-dependent complement activation. Jönsson, Göran; Lood, Christian; Gullstrand, Birgitta; Holmström, Eva M; Selander, Barbro; Braconier, Jean Henrik; Sturfelt, Gunnar; Bengtsson, Anders; Truedsson, Lennart Published in: Clinical Immunology DOI: 10.1016/j.clim.2012.06.008 2012 Link to publication Citation for published version (APA): Jönsson, G., Lood, C., Gullstrand, B., Holmström, E. M., Selander, B., Braconier, J. H., Sturfelt, G., Bengtsson, A., & Truedsson, L. (2012). Vaccination against encapsulated bacteria in hereditary C2 deficiency results in antibody response and opsonization due to antibody-dependent complement activation. Clinical Immunology, 144(3), 214-227. https://doi.org/10.1016/j.clim.2012.06.008 Total number of authors: 9 General rights Unless other specific re-use rights are stated the following general rights apply: Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Read more about Creative commons licenses: https://creativecommons.org/licenses/ Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. LUND UNIVERSITY PO Box 117 221 00 Lund +46 46-222 00 00 Download date: 18. Mar. 2023 Vaccination against encapsulated bacteria in hereditary C2 deficiency results in antibody response and opsonisation due to antibody-dependent complement activation Göran Jönsson, a, *, 1 Christian Lood, b, 1 Birgitta Gullstrand, b Eva Holmström, b Barbro Selander, b Jean Henrik Braconier, a Gunnar Sturfelt, c Anders A Bengtsson, c and Lennart Truedsson b a Department of Infectious Diseases Skåne University Hospital, Lund, Sweden b Department of Laboratory Medicine, Section of Microbiology, Immunology and Glycobiology, Lund University, Lund, Sweden c Department of Rheumatology, Skåne University Hospital, Lund, Sweden Running title: C2 DEFICIENCY, INFECTION, AND VACCINATION 1 These authors contributed equally to this work * Address correspondence and reprint requests to M.D., PhD. Göran Jönsson, Department of Infectious Diseases, Skåne University Hospital, SE-221 85 Lund, Sweden E-mail address: [email protected] Abbreviations used in this paper: C2D, hereditary C2 deficiency or C2-deficient, C4b2a, classical pathway C3 convertase, GMC, geometric mean concentration, Hib, Haemophilus influenzae type b, MBL, mannan-binding lectin or mannose-binding lectin, Neisseria meningitidis, N. meningitidis, Streptococcus pneumoniae, S. pneumoniae. 1 Abstract Hereditary C2 deficiency (C2D) is an important susceptibility factor for invasive infections caused by encapsulated bacteria such as pneumococci and Haemophilus influenzae type b. The infections are mostly seen in childhood indicating that antibody-mediated acquired immunity is affected. C2D persons and healthy controls were vaccinated with ActHIB® and Pneumo23®. Analysis of specific antibodies to pneumococci serotype 6B, 7F, and 23F, and Hib was performed. Post-vaccination IgG antibodies against pneumococci serotype 6B and 23F at a concentration ≥1.0 mg/L was found in similar frequency in C2D persons and controls. Post-vaccination sera from C2D persons showed poor complement-mediated opsonization and phagocytosis of pneumococci by granulocytes when depending on classical and lectin pathway activation only, but increased (p=0.007) and equaled that of the normal controls when also alternative pathway activation was allowed due to antibody-dependent C2 bypass activation. In conclusion, the C2D persons benefited from the vaccination and achieve an increased phagocytic capacity. Keywords The complement system, C2 deficiency, Invasive infection, Vaccination, Opsonization, Phagocytosis 2 1. Introduction Complement supports many immunological functions that contribute to protection from disease as well as to expression of disease manifestations. The complement system can be activated mainly through three pathways: the classical (C1qr s , C4 and C2), the alternative 2 2 (C3, factor B, factor D and properdin), and the lectin pathway (MBL or ficolins /MASPs, C4 and C2) [1]. Each of the three activation pathways leads to the formation of a C3 convertase, C4b2a for the classical and the lectin pathway and C3bBb for the alternative pathway. The C3 convertases cleave C3 which leads to formation of the principal opsonins C3b and iC3b that stimulate phagocytosis. Complement activation subsequently continues with the terminal complement components (C5-C9) that assemble to form a cell lysing membrane attack complex which may kill gram-negative bacteria such as Neisseria (N.) meningitidis and Haemophilus influenzae type b (Hib). However, gram-positive bacteria as for example Streptococcus (S.) pneumoniae resist the bactericidal action of C5-C9. Hereditary deficiency of the second component of complement (C2D) is one of the most common complement deficiency states in populations of Western descent and has an estimated prevalence of 1 in 20,000 [2, 3]. Two principal variants of C2D have been described [4, 5]. The predominant variant of C2D is type I (90%), which is caused by homozygosity for a 28-base pair deletion in the C2 gene resulting in a complete lack of C2 synthesis. This C2 deficiency gene is usually part of the major histocompatibility complex (MHC) haplotype HLA-B18, S042, DR2 [4, 5, 6]. C2D is associated with autoimmune diseases such as systemic lupus erythematosus (SLE) and with an increased susceptibility to infections caused by encapsulated bacteria such as S. pneumoniae and Hib [2, 3, 7, 8, 9]. C2D 3 may also be a risk factor for development of atherosclerosis [7]. However, many persons with C2D are apparently healthy [2, 7, 8]. Vaccination may be beneficial in complement-deficient patients [9]. For instance, favorable in vitro responses to tetravalent meningococcal vaccination have been demonstrated in properdin deficiency and in late-complement component deficiencies [10, 11, 12]. Vaccination of C2D patients has been considered to be advisable despite a lack of supportive data [2, 13]. In C2D, generation of C3 fragments by action of the classical pathway C3 convertase C4b2a, which is an important mechanism for recruitment of complement-mediated defense by specific antibodies, will not work [14, 15]. Also, the classical pathway is known to promote antibody responses to thymus-dependent antigens [16] and might influence responses to thymus- independent antigens [17] such as polysaccharides [18]. These circumstances suggest that vaccination responses in C2D are uncertain particularly against polysaccharide antigens. Nevertheless, we have reported for two C2D patients that anticapsular antibodies supported serum bactericidal reactions against N. meningitidis and Hib [19]. This raises the question if immunization with pneumococcal polysaccharides would promote opsonophagocytic killing of S. pneumoniae in C2D. We have described a cohort of 40 C2D patients with a high frequency of severe infections (57%) mainly caused by encapsulated bacteria [7]. In a follow-up study of 44 C2D persons, we found that the G2M*n/G2M*n genotype was associated with protection against severe infections suggesting the involvement of an immunoglobulin (Ig)-dependent defense mechanism [20]. 4 In the present study we have measured antibody responses following vaccination with the 23- valent pneumococcal vaccine Pneumo23® and Hib conjugate vaccine ActHIB® in C2D. The antibody responses were compared with those in a control group. We also investigated if immunization could promote opsonophagocytosis of S. pneumoniae in C2D. Issues regarding the clinical effect of vaccination were addressed in the C2D patients through review of medical records and a mailed questionnaire. 2. Materials and Methods 2. 1. Patients and controls Between 1977 and 2007, 49 persons with C2D were identified in clinical routine analysis at the Clinical Immunology unit, University Hospital of Lund, Sweden. Since the initiation of the present study in 1993, 25 C2D persons were enrolled and a written informed consent was obtained from each person. None of the C2D persons or controls was vaccinated with any pneumococcal vaccine or Hib vaccine before inclusion in the present study. Demographics and clinical manifestations of the vaccinated persons are shown in table 1. The distribution of gender was equal between the C2D persons (F: M, 16:9) and controls (F: M, 39:12). However, the C2D persons (median 41 years, range 2-63 years) were older than the control group (median 27 years, 16-61 years, p=0.02, Mann-Whitney U test). The participants received the 23-valent pneumococcal vaccine, which contains 25 μg of the following type-specific capsular polysaccharide: 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F (Pneumo23®, Sanofi Pasteur MSD, 5 S.N.C., Paris, France). The C2D persons were also vaccinated with Haemophilus type b conjugate vaccine, tetanus toxoid conjugate (ActHIB®, Sanofi Pasteur MSD). In 4 of the C2D persons vaccinated with Haemophilus type b conjugate vaccine, the pre-or post-vaccination blood samples were not technically handled in accord with the study protocol and therefore excluded from further analysis. A control group consisting of 51 healthy persons was also vaccinated with Pneumo23® and ActHIB®. The investigation was approved by the Lund University Ethics Committee (protocol LU 350-93). 2. 2. Follow-up of adverse reactions and side effects to vaccination Adverse reactions were followed among the C2D persons and controls. All vaccinated persons were asked to inform about any complaints by phone or by visiting the clinic during the first week after vaccination. A standardized form was filled out at three follow-up visits to the clinic (one month, 6 months and one year, respectively). No short-term adverse reactions as well as late adverse reactions and side effect were documented in the C2D persons. No SLE flare was triggered by the vaccination. Among controls, side effects were observed in one of the vaccinated. The person that experienced side effects to Pneumo23® in the control group was recorded for fever, swelling and redness at the site of vaccination. All symptoms resolved completely after three days. 2. 3. Assessment of invasive infections caused by pneumococci and Hib Medical records were reviewed and discussed with patient’s physicians. The investigation was supplemented with a mailed questionnaire. The following questions were addressed: Since the inclusion in the present study have you had any severe infection? If applicable, what type of infection/s were you treated for? Which years and where were you treated for the 6 infection/s? After inclusion in the present study have you had any additional vaccination? Year and name of the vaccine, alternatively what the vaccine should protect against? The questionnaire was given to 24 of the 25 C2D persons included in the study and they all responded. One C2D person died during the investigated period. The questionnaire was addressed to C2D persons parents if they were <18 years. 2. 4. Serum samples Venous blood samples were collected from all subjects before vaccination, and at 4 to 6 weeks after vaccine administration. In accord with a standardized protocol, serum samples were without delay stored in aliquots at −80°C until they were analyzed or used in the experiments. In 4 C2D persons serum samples were collected over 4-6 years for long-term follow-up after vaccination. Serum samples from 12 of the C2D persons, selected to cover the range of specific anti- pneumococcal antibodies were used in complement deposition and phagocytosis assays. An equally sized control group was used with sera matched according to antibody response. 2. 5. Antibodies to pneumococcal and Hib capsular polysaccharides Specific IgG, IgA, IgM, IgG1 and IgG2 to capsular polysaccharides of S. pneumoniae (serotypes 6B, 7F, and 23F) and Hib were determined by ELISA [21, 22, 23, ]. Hib was measured by antibodies to the capsular polysaccharide in human sera using an antigen composed of Haemophilus type b oligosaccharides conjugated to human serum albumin kindly provided by Dr. Moon H. Nahn (Departments of Microbiology and Pathology University of Alabama at Birmingham 845 19th Street South, BBRB614 Birmingham, 7 Alabama 35294). Purified pneumococcal capsular polysaccharides were provided by Pasteur Mérieux Connaught (Marcy-l’Etoilt, France). Pneumococcal C-polysaccharide for preabsorption of serum samples was purchased from Statens Serum Institut (Copenhagen, Denmark). In the assay, bound IgG, IgA and IgM was detected with corresponding goat anti- human IgG, IgA and IgM alkaline phosphatase conjugate (γ-, α-, and μ-chain specific F(ab’) 2 fragment, product no. A-3312 (IgG), A-3062 (IgA) and A-1067 (IgM) (Sigma Biosciences, St. Louis, MO, USA). Monoclonal anti-human IgG1 (NL-16) and anti-human IgG2 (HP6014) were from Skibio (Bedfordshire, UK). For detection of the monoclonal antibodies alkaline phosphatase conjugated goat anti-mouse IgG antibodies (Dako, Glostrup, Denmark) were used. The color reaction was developed with p-nitrophenylphosphate (1 mg/mL) in diethanolamine, pH 9.8, for one hour at room temperature. Absorbance was measured at 405 nm in a Multiscan Plus photometer (Labsystems Ltd., Helsinki, Finland). Values obtained were mean absorbance values from two coated wells with subtraction of background absorbance in the uncoated well. For expression of antibody concentrations in mg/L, calibration of a local reference serum was made against an international calibrator provided by Dr C Frasch, Bethesda, MD, USA (anti-pneumococcal antibodies lot 89 SF and anti-Hib antibodies serum pool lot 1983). The anti-Hib pool contained : IgG 60.9 mg/L, IgA 5.6 mg/L, IgM 3.5 mg/L, IgG1 30.9 mg/L, and IgG2 16.1 mg/L. The detection limits for the ELISA tests were calculated to be between 0.01 µg/L and 0.03 mg/L when the limit was defined as the lowest point on the dilution curve significantly higher than baseline (>+2 SD). Post- vaccination concentrations of specific IgG antibodies ≥1 mg/L were considered as long-term protective against infections caused by S. pneumoniae and Hib [24, 25, 26, 27]. All data concerning pre- and post-vaccination antibody concentrations are shown in the supplementary figures s1-s5. 8 2. 6. Immunoglobulins and complement proteins IgG, IgA, IgM, C3 and C4 were determined by turbidometry (Cobas Mira, Roche Diagnostic, Basel, Switzerland). Age-related immunoglobulin reference intervals were used [28, 29]. Concentrations of the IgG subclasses IgG1, IgG2 and IgG3 were determined by single immunodiffusion and 2.5-97.5 percentiles age-related reference intervals were used [30]. IgG4 levels were measured with a commercial ELISA (Bindazyme, The Binding Site Ltd, Birmingham, UK). Screening for detection of complement deficiency was mainly performed with hemolytic gel assays [31]. C3 and C4 were determined by turbidimetry (Cobas Mira, Roche Diagnostic). C2 concentrations were measured by electroimmunoassay and given in mg/L assuming a normal concentration at 26 mg/L [32]. C2D was defined as serum C2 concentration <0.5 mg/L. 2. 7. Preparation of S. pneumoniae serotype 23F Pneumococci serotype 23F, obtained from Statens Serum Institut, was stored on glass beads at -70°C. The pneumococci were cultured from the glass beads over night at 37°C anaerobically on blood agar and subcultured once before use. After inoculation into Todd-Hewitt broth, the bacteria were grown at 37°C until the absorbance was 0.4 at OD . The bacteria were washed 600 in 0.15 M NaCl and killed by addition of 0.8% glutaraldehyde in 0.15 M NaCl for 20 min. After additional washes the bacteria absorption was adjusted to 1.0 at OD and the bacteria 600 were frozen in small aliquots at -80°C. 2. 8. Analysis of opsonization of pneumococci by complement fragments 9