Development of a large scale human complement source for use in bacterial immunoassays

Journal of Immunological Methods 391 (2013) 39–49

Contents lists available at SciVerse ScienceDirect

Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim

Research paper

Development of a large scale human complement source for use in bacterial immunoassays Charlotte Brookes, Eeva Kuisma, Frances Alexander, Lauren Allen, Thomas Tipton, Sanjay Ram, Andrew Gorringe, Stephen Taylor ⁎ Health Protection Agency, Porton Down, Salisbury, SP4 0JG, UK

a r t i c l e

i n f o

Article history: Received 23 November 2012 Received in revised form 8 February 2013 Accepted 13 February 2013 Available online 26 February 2013 Keywords: Complement IgG-depletion Immunoassay Bactericidal Neisseria

a b s t r a c t The serum bactericidal assay is the correlate of protection for meningococcal disease but the use and comparison of functional immunological assays for the assessment of meningococcal vaccines is complicated by the sourcing of human complement. This is due to high levels of immunity in the population acquired through natural meningococcal carriage and means that many individuals must be screened to find donors with suitably low bactericidal titres against the target strain. The use of different donors for each meningococcal strain means that comparisons of assay responses between strains and between laboratories is difficult. We have developed a method for IgG-depletion of 300 ml batches of pooled human lepirudin-derived plasma using Protein G sepharose affinity chromatography that retains complement activity. However, IgG-depletion also removed C1q. This was also eluted from the affinity matrix, concentrated and added to the complement source. The final complement source retained mean alternative pathway activity of 96.8% and total haemolytic activity of 84.2% in four batches. Complement components C3, C5, properdin and factor H were retained following the process and the IgG-depleted complement was shown to be suitable for use in antibodymediated complement deposition and serum bactericidal activity assays against serogroup B meningococci. The generation of large IgG-depleted batches of pooled human plasma allows for the comparison of immunological responses to diverse meningococcal strain panels in large clinical trials. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The complement system is an important component of innate and adaptive immunity and is involved in the host response to invading microbes (Morgan et al., 2005; Dunkelberger and Song, 2010), and many immunoassays have been developed to measure complement-mediated immunity to a variety of bacterial pathogens (Romero-Steiner et al., 1997; Balmer and Borrow, 2004; Borrow et al., 2005; Henckaerts et al., 2006; Aase et al., 2007; Plested and Granoff, 2008; Guttormsen et al., 2009). The availability of a standard

⁎ Corresponding author. Tel.: +44 1980 6129963; fax: +44 1980 619936. E-mail address: [email protected] (S. Taylor). 0022-1759/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jim.2013.02.007

source of human plasma or serum for use as a complement source in immunoassays is a key issue for the assessment of vaccine induced immunity (Zollinger and Mandrell, 1983; Santos et al., 2001). Acquiring a human complement source for use in functional immunoassays to understand immunity to meningococcal disease has been complicated by the large amounts of naturally acquired cross-reactive antibodies in the human population which occur following nasopharyngeal carriage of N. meningitidis and commensal Neisseria strains, which has been observed in serological surveillance studies (Reller et al., 1973; Trotter et al., 2003, 2007, 2008, 2012). Thus plasma from these subjects can evoke meningococcal bacteriolysis in the absence of test antiserum and cause interference in the results of the immunoassays (Santos et al., 2001). A human complement source for use

40

C. Brookes et al. / Journal of Immunological Methods 391 (2013) 39–49

in immunoassays can be obtained from agammaglobulinaemic patients (Ala'Aldeen and Borriello, 1996). However, these individuals are rare in the population and are usually treated with immunoglobulin preparations (Kaveri et al., 2011). Alternatively, large numbers of volunteers can be screened to find a complement source which lacks intrinsic cross-reactive antibody to the test strain. The high prevalence of antimeningococcal antibodies makes these individuals rare and thus this often results in a different complement source from a different individual being used for each strain in assays, leading to poor inter-strain and inter-laboratory assay comparability. Complement is a key component for immunoassays assessing vaccine-induced immunity to meningococcal disease, with the pivotal role of complement in protection against meningococcal disease confirmed by the marked increase in disease susceptibility seen in complement deficient individuals (Figueroa and Densen, 1991; Lehner et al., 1992; Schneider et al., 2007; Hellerud et al., 2010). A central role for serum bactericidal activity (SBA) in the protection against meningococcal disease was established by Goldschneider et al. (1969a). This study described an inverse relationship between SBA titres obtained with a human complement source lacking intrinsic bactericidal activity and serogroup A, B and C disease by age. Thus whilst maternal antibodies were high disease incidence remained low, but when maternal antibody waned disease incidence increased. In addition, a bactericidal titre of ≥4 in military recruits sampled at the beginning of their training was associated with a lack of susceptibility to meningococcal disease (Goldschneider et al., 1969a, 1969b). In these studies human complement was used, but due to the difficulties in sourcing a human complement subsequent investigators have used baby rabbit complement as a substitute to improve standardisation of the SBA for evaluating polysaccharideinduced immunity, and this is recommended by the WHO for the assessment of meningococcal conjugate vaccines (World Health Organisation, 1976; Maslanka et al., 1997; Andrews et al., 2003; Keyserling et al., 2005; Kshirsagar et al., 2007; Gill et al., 2011). However, the use of rabbit complement results in increased bactericidal titres (Santos et al., 2001). Initial studies suggested that this was caused by the presence of low avidity anti-MenB capsular antibody; as absorption of these antibodies reduced bactericidal titres but they were still not comparable with titres achieved using human complement (Findlow et al., 2007). More recent work has shown that the increased bactericidal titres result from the specificity of meningococcal factor H binding protein for human factor H which leads to an overestimation of functional antibody activity when using baby rabbit complement (Granoff et al., 2009). In addition, meningococci have a further mechanism of complement factor H binding to NspA, which could also affect functional antibody activity (Lewis et al., 2010, 2012). Higher correlations have been observed in the SBA using rabbit (rSBA) or human complement (hSBA) for serogroup C meningococci, than serogroups A, W and Y. In these serogroups some samples which lack detectable bactericidal activity by hSBA have been shown to have protective bactericidal titres with rSBA (Findlow et al., 2009; Gill et al., 2011). This paper presents the development of an IgG-depleted human complement source that can be utilised in immunoassays to assess complement-mediated immunity to N. meningitidis. The method presented allows the depletion of plasma whilst

retaining key complement cascade components and total haemolytic and alternative pathway activities. 2. Material and methods 2.1. Collection of plasma or serum Volunteer blood was taken using either vacutainer tubes containing heparin (Becton Dickinson, UK), serum separator tubes with silica clot activator (Becton Dickinson, UK) or 50 μg/ml of the anti-coagulant lepirudin (Movianto, UK) (Sprong et al., 2004). Tubes were then centrifuged at 1000 g for 10 min to pellet the cells. The plasma, or serum was then removed and stored at − 80 °C. 2.2. Generation of sera Groups of ten 6–8-week-old BALB/c mice were immunised by subcutaneous injection with three doses of 10 μg N. meningitidis 44/76 outer membrane vesicles (OMVs) containing 0.33% Alhydrogel (Gorringe et al., 2005) at day 0, 21 and 28, before sera were collected on day 35. The human sera used were obtained from adult volunteers participating in a clinical trial of an experimental OMV vaccine based on N. lactamica (Gorringe et al., 2009). 2.3. Antibody depletion Pooled lepirudin-derived plasma from more than 10 adult volunteers was centrifuged at 1200 g (10 min, 4 °C) prior to applying to a 360 ml Protein G (GE) Sepharose column (5 × 18 cm, linear flow rate 0.5 cm/min) connected to an AKTA-FPLC (GE) equilibrated with PBS at 4 °C. Fractions were analysed by SDS PAGE (NuPage 4–12% Bis Tris gel, MOPS buffer, Invitrogen Life Sciences) and complement activity and component concentrations were analysed as described below. IgG-depleted plasma and C1q-containing fractions were concentrated in a dialysis membrane on a bed of polyethylene glycol (PEG) 20,000 (Sigma Aldrich). 2.4. Total haemolytic and alternative complement activity radial immunodiffusion assays Complement function was determined using total haemolytic and alternative pathway complement radial immunodiffusion assay kits (Binding Site, UK) according to the manufacturer's instructions. Briefly, lyophilised calibrator and control samples were reconstituted using distilled water, and all samples were kept pre-cooled on ice prior to the assay. 5 μl of test sample, calibrator dilutions or control samples were added to wells of either sheep erythrocyte agarose gel sensitised with rabbit anti-sheep erythrocyte antibody (total haemolytic assay) or chicken erythrocytes (alternative haemolytic assay). Radial diffusion occurred during overnight incubation at 4 °C before incubating at 37 °C for 45 min to allow the complement cascade to occur. The diameters of the lysis rings were measured, calibrator dilutions plotted and the complement activity calculated from the standard curve.

C. Brookes et al. / Journal of Immunological Methods 391 (2013) 39–49

2.5. Bacteria N. meningitidis 44/76 (B:15:P1.7,16/ST32) and M01240149 (B:4:P1.7-2,4/ST41) were used in the serum bactericidal assay or the antibody-mediated complement deposition assay. Bacteria used in the antibody-mediated complement deposition assay were killed for safe handling using a protocol developed to minimise chemical alteration of surface epitopes. After 4 h culture at 37 °C in 10 ml Frantz medium, meningococci were incubated with 0.2% (w/v) sodium azide and 17 μg ml−1 phenylmethylsulfonyl fluoride for 48 h at 37 °C. 2.6. Serum bactericidal assay A standard protocol was used to assess the bactericidal activity of test sera (Findlow et al., 2006) with the exception that bacteria were used following overnight growth on Columbia blood agar (bioMérieux) at 37 °C with 5% CO2, and used to inoculate 10 ml Frantz medium, and incubated at 37 °C with shaking for 3 h. Briefly, bacteria were resuspended in bactericidal buffer (BB), (Hanks buffered saline solution (Invitrogen) and 1% bovine serum albumin (BSA) (Sigma Aldrich)) before the OD600 nm was measured and bacteria were diluted in BB to 6 × 104 CFU/ml. 20 μl of heat inactivated serum was added to the first well, and was diluted across a microtitre plate using doubling dilutions. 10 μl of N. meningitidis were added to each well followed by 10 μl of human complement. The plate was then incubated at 37 °C for 1 h with shaking at 65 rpm. Each sample and control well was then plated out onto Columbia blood agar using the tilt method, air dried and incubated overnight at 37 °C with 5% CO2. The following day, colonies were counted and a titre assigned to the reciprocal dilution which gave >50% killing compared with the bacteria and complement control.

41

well and incubated for 1 h at room temperature and washed four times. 100 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution was added to each well and the plate left in the dark to develop for 30 min before the reaction was stopped using H2SO4. Sample absorbance was measured at 450 nm and the OD of the standard curve used to determine the sample concentration. 2.9. Complement component ELISAs Complement component ELISAs were performed using manufacturer's instructions (C1q and factor H, Hycult, UK; factor P, C3 and C5, USCNK Life Science Inc., China). In brief, 100 μl of test sample or assay standard was added to pre-coated wells, and then incubated at room temperature (20–25 °C) for 60 min for C1q and factor H, or at 37 °C for 2 h for factor P, C3 and C5. The plates were then washed with ELISA wash buffer and wells were incubated in 100 μl biotinylated detection antibody. The plate was then washed and 100 μl of streptavidin peroxidase conjugate added to all wells. Finally wells were incubated in TMB and concentration values determined as above. 2.10. Albumin concentration determined by IDEXX Plasma albumin concentration was determined using a ‘dry slide’ technology analyser (VetTest; IDEXX laboratories). 2.11. Statistics Statistical significance from ELISA and radial immunodiffusion assays were calculated using a two sample T-test. A significant difference of P b 0.01 is represented by **. Error bars represent the standard error of the mean (SEM). Flow cytometry overlay plots were analysed using the median fluorescence index (MFI).

2.7. Antibody-mediated C5b-9 or C3c deposition assay 3. Results Antibody-mediated C5b-9 or C3c deposition was evaluated as previously described (Martino et al., 2012). Briefly, 5 μl of test serum (heat inactivated) was added to 90 μl of target bacteria at an OD600 nm 0.1 in blocking buffer of 2% BSA in PBS followed by 5 μl of IgG-depleted human plasma and incubated for 45 min with shaking (900 rpm) at 37 °C. This was then centrifuged at 3060 g for 5 min and washed with blocking buffer and repeated twice before the addition of 200 μl FITC anti-human C3c (Abcam, UK) at 1/500 in blocking buffer and Alexafluor 647 nm anti human SC5b-9 (Quidel, USA) at 1/4000 in blocking buffer. This was incubated for 20 min at 4 °C, before being washed twice more with BB. The samples were then analysed by flow cytometry. 2.8. Total IgG, IgM, and IgA ELISAs IgG, IgM and IgA ELISAs were performed according to the manufacturer's instructions (Bethyl Laboratories). Briefly, 100 μl of, either standard, or assay sample was added to wells which had been precoated with coating antibody and incubated at room temperature (20–25 °C) for 1 h. The plate was then washed with ELISA wash buffer (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0) four times. 100 μl horseradish peroxidase-conjugated detection antibody was added to each

3.1. Comparison of complement activity in heparinised plasma, lepirudin plasma and serum Complement activity can be adversely affected by the use of some anti-coagulants (Mollnes et al., 2002; Amara et al., 2008), thus different plasma or serum collection methods were compared to determine the optimal complement source for use in immunoassays. Samples from a volunteer donor were collected either as serum or anticoagulated with lepirudin or heparin. These samples were compared in a total haemolytic complement activity RID and it was found that plasma collected into lepirudin had the greatest haemolytic activity (Fig. 1A). A visual comparison between the lysis circles from lepirudin or heparin anticoagulated plasma and serum following a total haemolytic RID demonstrated a double banded effect when using heparinised plasma (Fig. 1B). It has been shown previously that there is increased activation of complement factors in serum compared to lepirudin or heparin plasma (Mollnes et al., 2002), and while serum performed equally with lepirudin plasma for total haemolytic activity (Fig. 1A), less antibody-dependent deposition of membrane attack complex was seen on the surface of whole meningococci using serum as a complement source (data not shown). Plasma anti-coagulated

42

C. Brookes et al. / Journal of Immunological Methods 391 (2013) 39–49

A

B

Lepirudin

Heparin

Serum

Fig. 1. Total haemolytic complement activity measured by radial immunodiffusion assay comparing complement activity from plasma anti-coagulated with lepirudin or heparin with serum (A) and photos of the lysis circles for each condition tested (B).

with lepirudin was thus chosen for the further development of an IgG-depleted complement source. 3.2. Protein G affinity chromatography to remove IgG from the plasma In order to produce a complement source which would not contain intrinsic bactericidal activity, Protein G Sepharose affinity chromatography was performed to remove IgG from plasma collected by volunteer donation. The chromatography profile (Fig. 2) and the fractions containing total haemolytic and

UV Absorbances (mAU)

mAU

alternative pathway activity were identified. These fractions were pooled and concentrated to 90% of the original plasma volume by dialysis against PEG 20,000. The restoration of the original volume was confirmed by analysis of albumin concentration in the plasma (Fig. 3). 3.3. Recovery of C1q for addition to IgG-depleted plasma Evaluation of IgG-depleted plasma by C1q ELISA showed a significant (p b 0.01) reduction in C1q during the affinity chromatography (Fig. 4A). Elution of C1q was then performed as previously described by Kolb et al. (1979) using a NaCl gradient (Fig. 4B). The presence of C1q in the eluted fractions

400

300

200

∗∗

Fractions Pooled

100

0 0

125

250

375

500

625

750 ml

Volume (ml) Fig. 2. Chromatogram illustrating the depletion of IgG from plasma and the combination of fractions selected for pooling on the basis of complement activity.

Fig. 3. Concentration of albumin was determined by IDEXX (**p b 0.01 by T-test). Bars represent the mean levels of albumin for the four depletion batches evaluated.

C. Brookes et al. / Journal of Immunological Methods 391 (2013) 39–49

(Fig. 4C) was confirmed by C1q ELISA (Fig. 4D) and fractions collected and confirmed as positive for C1q were pooled and concentrated to one tenth of the original plasma volume by dialysis against PEG 20,000 and then added to the concentrated flow through pool to restore the C1q concentration (Fig 4A).

3.4. Evaluation of antibody concentration following Protein G Sepharose chromatography Removal of IgG was confirmed by total IgG ELISA which demonstrated removal of IgG to undetectable levels (assay lower limit of detection 0.69 ng/ml) (Table 1). Significant decreases in IgM were also observed following IgG depletion and IgA levels remained unchanged in comparison with pre-depletion samples (Table 1).

43

3.5. Comparison of complement cascade activity in four batches of IgG-depleted plasma Following IgG-depletion, PEG concentration and addition of fractions containing eluted C1q, the plasma complement activity was evaluated by using total haemolytic and alternative pathway RID assays. Four batches were investigated to assess the process reproducibility, three batches were prepared from the same pool of volunteer plasma and batch 4 was from a different volunteer pool. Depletion batch four therefore was used to evaluate the differences between two plasma pools. Total haemolytic complement activity of the final preparation was found to be between 75.6% and 91.3% of the pre-depletion values (Fig. 5A). Alternative pathway complement activity following IgG-depletion was between 91.4% and 105% (Fig. 5B).

Fig. 4. A, C1q ELISA comparing C1q concentration in pre-depletion native plasma with IgG depleted plasma, PEG concentrated IgG depleted plasma and final product complement source with C1q fractions added (**p b 0.01 by T-test). Bars represent the mean levels of C1q for the four depletion batches evaluated. B, Chromatogram showing elution of C1q from column using an NaCl gradient at 22 °C. C, Enlarged view of the profile of fractions containing C1q. D, 15 ml fractions were collected starting at 538 ml and the presence of C1q determined by ELISA.

44

C. Brookes et al. / Journal of Immunological Methods 391 (2013) 39–49

Fig. 4 (continued).

3.6. Effect of IgG-depletion on complement components properdin, factor H, C3, C5

3.7. Antibody-mediated C5b-9 and C3c deposition on N. meningitidis comparing native and IgG-depleted plasma

The complement system is composed of at least 35 fluid phase and membrane bound proteins and several central components were chosen for evaluation by ELISA of the IgG-depleted plasma. No significant difference in levels of properdin, factor H or C3 were found between pre-depletion plasma, PEG-concentrated IgG-depleted plasma or the final IgG-depleted complement source which had added C1q (Fig. 6A, B and C). No significant difference was observed in the C5 concentration between pre-depletion and final product (Fig. 6D) but a significant drop in C5 was observed in the PEG-concentrated IgG-depleted plasma, indicating that this component was retained on the column and co-eluted with the C1q. The presence of C5 was confirmed in C1q fractions by ELISA (data not shown).

Antibody-mediated C3c and C5b-9 deposition was compared using mouse anti-N. meningitidis OMV serum and either pooled native plasma or IgG-depleted final product plasma. Native plasma demonstrated high levels of intrinsic complement activating antibody with levels of C3c deposition higher in the complement-only control (MFI = 357) than in the mouse positive control OMV test serum (MFI = 199) (Fig. 7A). IgG depletion removed the high levels of C3c deposition in the complement-only control (MFI = 128) allowing the measurement of the complement deposition mediated by the mouse anti-OMV test serum (MFI = 199) (Fig. 7B). Levels of antibodymediated C5b-9 deposition were also shown to be higher using the native plasma complement only control (MFI = 34) and IgG-depletion greatly reduced control C5b-9 binding (MFI = 14) (Fig. 7C and D).

Table 1 Total IgG, IgM and IgA determined by ELISA comparing batches 1–4.

Native plasma Depletion batch Native plasma Depletion batch Native plasma Depletion batch Native plasma Depletion batch

1

IgG mg/ml (SEM)

IgM mg/ml (SEM)

IgA mg/ml (SEM)

3.43 (0.16) 0⁎⁎

1.05 0.74 1.03 0.92 0.88 0.65 0.96 0.77

2.84 2.44 3.11 2.86 2.52 2.13 2.53 2.78

3

3.50 (0.26) 0⁎⁎ 2.17 (0.08) 0⁎⁎

4

2.59 (0.05) 0⁎⁎

2

⁎⁎ p b 0.01,⁎ p b 0.05 by T-test.

(0.02) (0.02)⁎⁎ (0.08) (0.03)⁎⁎ (0.02) (0.03)⁎⁎ (0.06) (0.03)⁎⁎

(0.23) (0.17) (0.15) (0.20) (0.15) (0.09)* (0.12) (0.47)

3.8. Comparison of serum bactericidal assay titres using native or IgG-depleted plasma as the complement source A panel of fifteen human sera and a mouse serum raised against N. meningitidis 44/76 OMVs were tested for bactericidal activity with N. meningitidis 44/76 as the target strain using native or IgG-depleted plasma as the complement source. Titres obtained with the different complement sources were either the same or within one dilution (Table 2) which is considered assay variation. There were two exceptions to this with human samples 8 and 10, where titres obtained using IgG-depleted plasma were two dilutions lower than the titres obtained with native plasma.

C. Brookes et al. / Journal of Immunological Methods 391 (2013) 39–49

45

Fig. 5. Total haemolytic complement activity RID (A) and alternative pathway haemolytic activity RID (B) comparing the % haemolytic activity retained in the final complement source for four depletion batches. Depletion batches 1–3 are from the same pool of plasma collected from volunteers, depletion batch 4 originated from a separate pool of plasma from different volunteers included to evaluate the process consistency between plasma pools.

4. Discussion Complement is a key component in immunoassays evaluating vaccine-induced immunity to meningococcal disease

(Goldschneider et al., 1969a). Identifying human plasma/ serum which can be used as a complement source which lacks intrinsic bactericidal antibodies is complicated by the high levels of nasopharyngeal carriage of Neisseria species. Thus this study

Fig. 6. Comparison of complement component concentrations in pre-depletion native plasma with PEG concentrated IgG-depleted plasma and final product complement source with C1q fractions added for properdin (A); factor H (B); C3 (C) and C5 (D). Bars represent the mean levels of complement component for the four depletion batches evaluated (**p b 0.01 by T-test).

46

C. Brookes et al. / Journal of Immunological Methods 391 (2013) 39–49

A

B 199

199 5

5

357

128

C

D 3

3

254

34 211

14

Fig. 7. Flow cytometry overlay plot illustrating the levels of C3c deposition (A and B) and C5b-9 deposition (C and D) of a bacteria only control (solid black), complement only control (dashed black line) and M01240149 positive control mouse sera (solid black line) with either pre-depletion native plasma (A and C) or IgG-depleted final product complement source (B and D). Median fluorescence index (MFI) is presented above each peak.

describes a method to IgG-deplete large volumes (300 ml) of volunteer human plasma whilst retaining functional complement activity. Availability of large volumes of plasma which could be used as a complement source would allow for better comparability of SBA titres obtained with different strains and allow inter-laboratory standardisation of SBA results. Other researchers have used a variety of different human complement sources in immunoassays including serum, heparinised plasma and lepirudin-anticoagulated plasma (Zollinger and Mandrell, 1983; Mollnes et al., 2002; Sprong et al., 2004; Aase et al., 2007; Granoff et al., 2009). Initially, we evaluated differences between these complement sources from the same volunteer by determining the total haemolytic complement activity. Heparinised plasma had a defective but functioning complement cascade which was indicated by the presence of a double ring in the total haemolytic RID (Fig. 1B).

The exact mechanism of dysfunction to the complement cascade in heparinised plasma is unknown, but heparin has been shown to have binding sites for many different complement components which could have contributed (Sahu and Pangburn, 1993; Blackmore et al., 1996, 1998). Lepirudin-derived plasma showed the greatest total haemolytic activity (Fig. 1) and has been used as an anticoagulant in other studies (Sprong et al., 2004) due to the low levels of complement activation (Mollnes et al., 2002). Additionally, the use of serum as a complement source has been shown to lead to less antibody-dependent deposition of C5b-9 membrane attack complex onto the surface of whole meningococci, compared to lepirudin plasma (data not shown). Lepirudin-anticoagulated plasma was therefore selected for all further process development. IgG-depleted serum has previously been used as a source of complement in immunoassays (Aase et al., 2007; Ray et al.,

C. Brookes et al. / Journal of Immunological Methods 391 (2013) 39–49 Table 2 Serum bactericidal assay titres obtained with N. meningitidis 44/76, comparing titres achieved with pre-depletion native plasma with IgG-depleted final product complement source with a panel of adult human sera and a N. meningitidis 44/76 mouse positive control serum generated by immunisation of mice with 44/76 OMVs. Sera

Native plasma

Depletion batch 1

44/76 OMV mouse Human 1 Human 2 Human 3 Human 4 Human 5 Human 6 Human 7 Human 8 Human 9 Human 10 Human 11 Human 12 Human 13 Human 14 Human 15

2048 32 32 128 32 64 128 16 1024 128 32 32 32 32 32 512

1024 64 16 128 32 64 64 32 256 64 8 64 64 16 32 256

2011). Small volumes of IgG-depleted complement (1–2 ml) have been prepared on a Protein G column immediately prior to use in immunoassays (Aase et al., 2007; Ray et al., 2011; Giuntini et al., 2012a, 2012b). Small volume depletion has the disadvantage of introducing potential day-to-day variation between the depleted plasma used as each depletion cannot be extensively evaluated on each day for total haemolytic and alternative pathway complement activities, removal of key complement components and the dilution effect of the process, which might be variable in a small scale process. While it has been shown to be possible to account for some of the variation in activity after depletion, by measuring the total haemolytic activity and adjusting the volume of complement in the assays accordingly (Beernink et al., 2011), the use of a larger scale process, as described here, allows batches to be thoroughly characterised and frozen at −80 °C in 1 ml single use aliquots for use in studies such as clinical trials. Protein G Sepharose was chosen as it was able to completely remove IgG from the plasma compared to either Protein A or Protein L Sepharose (data not shown). Total antibody ELISA demonstrated the effective removal of IgG to below detectable levels (lower assay limit 0.69 ng/ml) which was expected due to the high affinity of Protein G for IgG. Significant (P b 0.05) reductions in the levels of total IgM were also observed which was an unexpected finding as Protein G does not bind IgM (Bjorck et al., 1984). No significant difference was observed following chromatography in levels of IgA and as IgA is not able to initiate complement-mediated bactericidal activity (Vidarsson et al., 2001), it was considered that there would be little effect of IgA levels remaining high in the final product. However, IgM is able to interact with C1q to initiate complement activation (Kojouharova et al., 2010) and further work might investigate the potential for IgM removal and its effect on the final complement source. We investigated the levels of C1q before and after affinity chromatography with Protein G Sepharose and showed that C1q was removed in the process of IgG depletion. Removal of C1q was thought to be due to the activation of C1q to IgG

47

bound to Protein G Sepharose (Bjorck and Kronvall, 1984; Tao et al., 1991). A further NaCl gradient elution step was included to remove C1q from the affinity matrix as previously described for purification of C1q (Kolb et al., 1979). C1q was identified in fractions using a C1q ELISA (Fig. 4C and D), concentrated and added back into the final IgG-depleted plasma. C1q ELISA confirmed that C1q levels were restored (Fig. 4A). Functional analysis of the final complement source confirmed that the total haemolytic and alternative pathway complement activities had also been restored (Fig. 5). It was also identified that complement component C5 was removed during the IgG-depletion (Fig. 6D). This finding was unexpected but was consistent in all batches that were tested. However, C5 was eluted from the Protein G Sepharose column during the salt gradient to elute C1q and was identified in the same fraction pools by C5 ELISA and was therefore restored to pre-depletion concentrations when the C1q-containing fraction pool was added back to the final complement source (Fig. 6D). Evaluation of the depletion effect on levels of properdin, factor H and C3 was conducted by ELISA with no significant effect seen on these components of the cascade. There are however other complement components which were not specifically evaluated by ELISA, which may have been depleted during the processing. Consistency between large scale depletion batches was assessed in this study. Depletion batches 1–3 were prepared from the same plasma pool obtained from >10 volunteers, whereas depletion batch 4 was from a second pool of plasma obtained from >10 volunteers. Total haemolytic and alternative pathway complement activities following depletion were found to be consistent between all batches (Fig. 4). Complement component concentrations as evaluated by ELISA, also showed similar levels between depleted batches (Fig. 6). IgG was undetectable following depletion in all batches of depleted plasma (Table 1). This indicated that the process described produces a consistent product within and between pools of volunteer plasma. A direct comparison was made between SBA titres obtained against N. meningitidis strain 44/76 with plasma from a volunteer who was naturally low in bactericidal activity, and final product IgG depleted plasma with a panel of 15 human sera and a mouse anti-OMV serum. All samples were assigned titres that were either the same or within one dilution with the exception of two sera (Table 2), leading to the conclusion that the depleted complement was suitable for use as a complement source in bactericidal assays against serogroup B meningococci. IgG-depleted plasma was also used in assays to assess antibody-mediated C3c and C5b-9 deposition on the surface of N. meningitidis M01240149. A comparison was made between native plasma and IgG-depleted plasma with antibody mediated C3c and C5b-9 deposition with anti OMV mouse sera raised against M01240149 (Fig. 7A–D). Complement only control assay backgrounds were found to be greater than antibody mediated C3c deposition of anti OMV mouse sera raised against M01240149 when using native plasma, complement only control C3c deposition levels were reduced when using IgG-depleted complement (Fig. 7A and B). C5b-9 deposition levels in the complement only control were reduced by approximately 50% following depletion (Fig. 7C and D). Use of the IgG-depleted complement source in an antibody mediated C3c and C5b-9 deposition assay showed that this

48

C. Brookes et al. / Journal of Immunological Methods 391 (2013) 39–49

complement source could be used in other types of immunoassays in addition to the SBA. We have evaluated the final complement source for use in immunoassays using serogroup B meningococci. However, the developed IgG-depleted complement source could also be used to evaluate other serogroups of meningococci and also have further applications for evaluating complement-mediated immunity with other bacterial pathogens where complementmediated immunity is thought to be important. Other researchers have used rabbit complement to evaluate complement-mediated immune responses to meningococcal disease due to the difficulty in acquiring a human complement source which is low in intrinsic bactericidal activity (Maslanka et al., 1997), and rabbit complement has been used for licensure of conjugate polysaccharide vaccines (Jodar et al., 2000). However, use of rabbit complement results in elevated bactericidal titres (Zollinger and Mandrell, 1983; Santos et al., 2001; Gill et al., 2011). These elevated titres are now known to be attributable to the specificity of meningococcal factor H binding protein for human factor H (Granoff et al., 2009), and data correlating rabbit and human complement bactericidal titres with serogroups A, W and Y showed a poor correlation, indicating that it is not just an elevated response with rabbit complement (Gill et al., 2011), other complement factors may also have a species specific effect which has yet not been established. This emphasises the importance of evaluating complement-mediated immune responses with a human complement source that can be used to evaluate any strain due to low levels of bactericidal activity. This study has developed a method for IgG depletion of human plasma, which successfully removes IgG and retains total haemolytic and alternative haemolytic complement activity. Good consistency between depletion batches and suitability for use in antibody-mediated complement binding and SBA assays has been demonstrated. Thus the use of this complement source could be used to improve assay standardisation and allow improved comparison of immune responses across bacterial strains. Acknowledgements This study was funded by the UK National Institute for Health Research. Sanjay Ram was supported by grants AI054544, AI084048 and AI32725 from the National Institute of Allergy and Infectious Diseases, NIH. Special thanks to Professor Paul Morgan and Dr. Claire Harris for their help and advice on this project. References Aase, A., Herstad, T.K., Merino, S., Brandsdal, K.T., Berdal, B.P., Aleksandersen, E.M., Aaberge, I.S., 2007. Opsonophagocytic activity and other serological indications of Bordetella pertussis infection in military recruits in Norway. Clin. Vaccine Immunol. 14, 855. Ala'Aldeen, D.A., Borriello, S.P., 1996. The meningococcal transferrin-binding proteins 1 and 2 are both surface exposed and generate bactericidal antibodies capable of killing homologous and heterologous strains. Vaccine 14, 49. Amara, U., Rittirsch, D., Flierl, M., Bruckner, U., Klos, A., Gebhard, F., Lambris, J.D., Huber-Lang, M., 2008. Interaction between the coagulation and complement system. Adv. Exp. Med. Biol. 632, 71. Andrews, N., Borrow, R., Miller, E., 2003. Validation of serological correlate of protection for meningococcal C conjugate vaccine by using efficacy

estimates from postlicensure surveillance in England. Clin. Diagn. Lab. Immunol. 10, 780. Balmer, P., Borrow, R., 2004. Serologic correlates of protection for evaluating the response to meningococcal vaccines. Expert Rev. Vaccines 3, 77. Beernink, P.T., Shaughnessy, J., Braga, E.M., Liu, Q., Rice, P.A., Ram, S., Granoff, D.M., 2011. A meningococcal factor H binding protein mutant that eliminates factor H binding enhances protective antibody responses to vaccination. J. Immunol. 186, 3606. Bjorck, L., Kronvall, G., 1984. Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J. Immunol. 133, 969. Bjorck, L., Miorner, H., Kuhnemund, O., Kronvall, G., Sundler, R., 1984. On the interaction between beta 2-microglobulin and group A streptococci. Scand. J. Immunol. 20, 69. Blackmore, T.K., Sadlon, T.A., Ward, H.M., Lublin, D.M., Gordon, D.L., 1996. Identification of a heparin binding domain in the seventh short consensus repeat of complement factor H. J. Immunol. 157, 5422. Blackmore, T.K., Hellwage, J., Sadlon, T.A., Higgs, N., Zipfel, P.F., Ward, H.M., Gordon, D.L., 1998. Identification of the second heparin-binding domain in human complement factor H. J. Immunol. 160, 3342. Borrow, R., Aaberge, I.S., Santos, G.F., Eudey, T.L., Oster, P., Glennie, A., Findlow, J., Hoiby, E.A., Rosenqvist, E., Balmer, P., Martin, D., 2005. Interlaboratory standardization of the measurement of serum bactericidal activity by using human complement against meningococcal serogroup b, strain 44/76-SL, before and after vaccination with the Norwegian MenBvac outer membrane vesicle vaccine. Clin. Diagn. Lab. Immunol. 12, 970. Dunkelberger, J.R., Song, W.C., 2010. Complement and its role in innate and adaptive immune responses. Cell Res. 20, 34. Figueroa, J.E., Densen, P., 1991. Infectious diseases associated with complement deficiencies. Clin. Microbiol. Rev. 4, 359. Findlow, J., Taylor, S., Aase, A., Horton, R., Heyderman, R., Southern, J., Andrews, N., Barchha, R., Harrison, E., Lowe, A., Boxer, E., Heaton, C., Balmer, P., Kaczmarski, E., Oster, P., Gorringe, A., Borrow, R., Miller, E., 2006. Comparison and correlation of Neisseria meningitidis serogroup B immunologic assay results and human antibody responses following three doses of the Norwegian meningococcal outer membrane vesicle vaccine MenBvac. Infect. Immun. 74, 4557. Findlow, J., Holland, A., Martin, D., Oster, P., Balmer, P., Borrow, R., 2007. Inadequacy of colominic acid as an absorbent intended to facilitate use of complement-preserved baby rabbit serum in the Neisseria meningitidis serogroup B serum bactericidal antibody assay. Clin. Vaccine Immunol. 14, 556. Findlow, H., Plikaytis, B.D., Aase, A., Bash, M.C., Chadha, H., Elie, C., Laher, G., Martinez, J., Herstad, T., Newton, E., Viviani, S., Papaspyridis, C., Kulkarni, P., Wilding, M., Preziosi, M.P., Marchetti, E., Hassan-King, M., La Force, F.M., Carlone, G., Borrow, R., 2009. Investigation of different group A immunoassays following one dose of meningococcal group A conjugate vaccine or A/C polysaccharide vaccine in adults. Clin. Vaccine Immunol. 16, 969. Gill, C.J., Ram, S., Welsch, J.A., DeTora, L., Anemona, A., 2011. Correlation between serum bactericidal activity against Neisseria meningitidis serogroups A, C, W-135 and Y measured using human versus rabbit serum as the complement source. Vaccine 30, 29. Giuntini, S., Beernink, P.T., Reason, D.C., Granoff, D.M., 2012a. Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity. PLoS One 7, e34272. Giuntini, S., Reason, D.C., Granoff, D.M., 2012b. Combined roles of human IgG subclass, alternative complement pathway activation, and epitope density in the bactericidal activity of antibodies to meningococcal factor h binding protein. Infect. Immun. 80, 187. Goldschneider, I., Gotschlich, E.C., Artenstein, M.S., 1969a. Human immunity to the meningococcus. I. The role of humoral antibodies. J. Exp. Med. 129, 1307. Goldschneider, I., Gotschlich, E.C., Artenstein, M.S., 1969b. Human immunity to the meningococcus. II. Development of natural immunity. J. Exp. Med. 129, 1327. Gorringe, A.R., Reddin, K.M., Funnell, S.G., Johansson, L., Rytkonen, A., Jonsson, A.B., 2005. Experimental disease models for the assessment of meningococcal vaccines. Vaccine 23, 2214. Gorringe, A.R., Taylor, S., Brookes, C., Matheson, M., Finney, M., Kerr, M., Hudson, M., Findlow, J., Borrow, R., Andrews, N., Kafatos, G., Evans, C.M., Read, R.C., 2009. Phase I safety and immunogenicity study of a candidate meningococcal disease vaccine based on Neisseria lactamica outer membrane vesicles. Clin. Vaccine Immunol. 16, 1113. Granoff, D.M., Welsch, J.A., Ram, S., 2009. Binding of complement factor H (fH) to Neisseria meningitidis is specific for human fH and inhibits complement activation by rat and rabbit sera. Infect. Immun. 77, 764. Guttormsen, H.K., Mascuch, S.J., West, J.C., Paoletti, L.C., 2009. A fluorescencebased opsonophagocytosis assay to measure the functional activity of antibody to group B Streptococcus. Hum. Vaccin. 5, 461.

C. Brookes et al. / Journal of Immunological Methods 391 (2013) 39–49 Hellerud, B.C., Aase, A., Herstad, T.K., Naess, L.M., Kristiansen, L.H., Troseid, A.M., Harboe, M., Lappegard, K.T., Brandtzaeg, P., Hoiby, E.A., Mollnes, T.E., 2010. Critical roles of complement and antibodies in host defense mechanisms against Neisseria meningitidis as revealed by human complement genetic deficiencies. Infect. Immun. 78, 802. Henckaerts, I., Durant, N., De Grave, D., Schuerman, L., Poolman, J., 2006. Validation of a routine opsonophagocytosis assay to predict invasive pneumococcal disease efficacy of conjugate vaccine in children. Vaccine 25, 2518. Jodar, L., Cartwright, K., Feavers, I.M., 2000. Standardisation and validation of serological assays for the evaluation of immune responses to Neisseria meningitidis serogroup A and C vaccines. Biologicals 28, 193. Kaveri, S.V., Maddur, M.S., Hegde, P., Lacroix-Desmazes, S., Bayry, J., 2011. Intravenous immunoglobulins in immunodeficiencies: more than mere replacement therapy. Clin. Exp. Immunol. 164 (Suppl. 2), 2. Keyserling, H., Papa, T., Koranyi, K., Ryall, R., Bassily, E., Bybel, M.J., Sullivan, K., Gilmet, G., Reinhardt, A., 2005. Safety, immunogenicity, and immune memory of a novel meningococcal (groups A, C, Y, and W-135) polysaccharide diphtheria toxoid conjugate vaccine (MCV-4) in healthy adolescents. Arch. Pediatr. Adolesc. Med. 159, 907. Kojouharova, M., Reid, K., Gadjeva, M., 2010. New insights into the molecular mechanisms of classical complement activation. Mol. Immunol. 47, 2154. Kolb, W.P., Kolb, L.M., Podack, E.R., 1979. C1q: isolation from human serum in high yield by affinity chromatography and development of a highly sensitive hemolytic assay. J. Immunol. 122, 2103. Kshirsagar, N., Mur, N., Thatte, U., Gogtay, N., Viviani, S., Preziosi, M.P., Elie, C., Findlow, H., Carlone, G., Borrow, R., Parulekar, V., Plikaytis, B., Kulkarni, P., Imbault, N., LaForce, F.M., 2007. Safety, immunogenicity, and antibody persistence of a new meningococcal group A conjugate vaccine in healthy Indian adults. Vaccine 25 (Suppl. 1), A101. Lehner, P.J., Davies, K.A., Walport, M.J., Cope, A.P., Wurzner, R., Orren, A., Morgan, B.P., Cohen, J., 1992. Meningococcal septicaemia in a C6deficient patient and effects of plasma transfusion on lipopolysaccharide release. Lancet 340, 1379. Lewis, L.A., Ngampasutadol, J., Wallace, R., Reid, J.E., Vogel, U., Ram, S., 2010. The meningococcal vaccine candidate neisserial surface protein A (NspA) binds to factor H and enhances meningococcal resistance to complement. PLoS Pathog. 6, e1001027. Lewis, L.A., Carter, M., Ram, S., 2012. The relative roles of factor H binding protein, neisserial surface protein A, and lipooligosaccharide sialylation in regulation of the alternative pathway of complement on meningococci. J. Immunol. 188, 5063. Martino, A., Magagnoli, C., De Conciliis, G., D'Ascenzi, S., Forster, M.J., Allen, L., Brookes, C., Taylor, S., Bai, X., Findlow, J., Feavers, I.M., Rodger, A., Bolgiano, B., 2012. Structural characterisation, stability and antibody recognition of chimeric NHBA-GNA1030: an investigational vaccine component against Neisseria meningitidis. Vaccine 30, 1330. Maslanka, S.E., Gheesling, L.L., Libutti, D.E., Donaldson, K.B., Harakeh, H.S., Dykes, J.K., Arhin, F.F., Devi, S.J., Frasch, C.E., Huang, J.C., KrizKuzemenska, P., Lemmon, R.D., Lorange, M., Peeters, C.C., Quataert, S., Tai, J.Y., Carlone, G.M., 1997. Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. The Multilaboratory Study Group. Clin. Diagn. Lab. Immunol. 4, 156. Mollnes, T.E., Brekke, O.L., Fung, M., Fure, H., Christiansen, D., Bergseth, G., Videm, V., Lappegard, K.T., Kohl, J., Lambris, J.D., 2002. Essential role of the C5a receptor in E coli-induced oxidative burst and phagocytosis

49

revealed by a novel lepirudin-based human whole blood model of inflammation. Blood 100, 1869. Morgan, B.P., Marchbank, K.J., Longhi, M.P., Harris, C.L., Gallimore, A.M., 2005. Complement: central to innate immunity and bridging to adaptive responses. Immunol. Lett. 97, 171. Plested, J.S., Granoff, D.M., 2008. Vaccine-induced opsonophagocytic immunity to Neisseria meningitidis group B. Clin. Vaccine Immunol. 15, 799. Ray, T.D., Lewis, L.A., Gulati, S., Rice, P.A., Ram, S., 2011. Novel blocking human IgG directed against the pentapeptide repeat motifs of Neisseria meningitidis Lip/H.8 and Laz lipoproteins. J. Immunol. 186, 4881. Reller, L.B., MacGregor, R.R., Beaty, H.N., 1973. Bactericidal antibody after colonization with Neisseria meningitidis. J. Infect. Dis. 127, 56. Romero-Steiner, S., Libutti, D., Pais, L.B., Dykes, J., Anderson, P., Whitin, J.C., Keyserling, H.L., Carlone, G.M., 1997. Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells. Clin. Diagn. Lab. Immunol. 4, 415. Sahu, A., Pangburn, M.K., 1993. Identification of multiple sites of interaction between heparin and the complement system. Mol. Immunol. 30, 679. Santos, G.F., Deck, R.R., Donnelly, J., Blackwelder, W., Granoff, D.M., 2001. Importance of complement source in measuring meningococcal bactericidal titers. Clin. Diagn. Lab. Immunol. 8, 616. Schneider, M.C., Exley, R.M., Ram, S., Sim, R.B., Tang, C.M., 2007. Interactions between Neisseria meningitidis and the complement system. Trends Microbiol. 15, 233. Sprong, T., Moller, A.S., Bjerre, A., Wedege, E., Kierulf, P., van der Meer, J.W., Brandtzaeg, P., van Deuren, M., Mollnes, T.E., 2004. Complement activation and complement-dependent inflammation by Neisseria meningitidis are independent of lipopolysaccharide. Infect. Immun. 72, 3344. Tao, M.H., Canfield, S.M., Morrison, S.L., 1991. The differential ability of human IgG1 and IgG4 to activate complement is determined by the COOH-terminal sequence of the CH2 domain. J. Exp. Med. 173, 1025. Trotter, C., Borrow, R., Andrews, N., Miller, E., 2003. Seroprevalence of meningococcal serogroup C bactericidal antibody in England and Wales in the pre-vaccination era. Vaccine 21, 1094. Trotter, C., Findlow, J., Balmer, P., Holland, A., Barchha, R., Hamer, N., Andrews, N., Miller, E., Borrow, R., 2007. Seroprevalence of bactericidal and anti-outer membrane vesicle antibody to Neisseria meningitidis group B in England. Clin. Vaccine Immunol. 14, 863. Trotter, C.L., Borrow, R., Findlow, J., Holland, A., Frankland, S., Andrews, N.J., Miller, E., 2008. Seroprevalence of antibodies against serogroup C meningococci in England in the postvaccination era. Clin. Vaccine Immunol. 15, 1694. Trotter, C.L., Findlow, H., Borrow, R., 2012. Seroprevalence of serum bactericidal antibodies against group W135 and Y meningococci in England in 2009. Clin. Vaccine Immunol. 19, 219. Vidarsson, G., van Der Pol, W.L., van Den Elsen, J.M., Vile, H., Jansen, M., Duijs, J., Morton, H.C., Boel, E., Daha, M.R., Corthesy, B., van De Winkel, J.G., 2001. Activity of human IgG and IgA subclasses in immune defense against Neisseria meningitidis serogroup B. J. Immunol. 166, 6250. World Health Organisation, W., 1976. Requirements for meningococcal polysaccharide vaccine (requirements for biological substances no. 23). World Health Organ. Tech. Rep. Ser. 594, 72. Zollinger, W.D., Mandrell, R.E., 1983. Importance of complement source in bactericidal activity of human antibody and murine monoclonal antibody to meningococcal group B polysaccharide. Infect. Immun. 40, 257.