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Flow cytometry: An alternative method for direct quantification of antigens adsorbed to aluminum hydroxide adjuvant
Analytical Biochemistry 418 (2011) 224–230
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Flow cytometry: An alternative method for direct quantification of antigens adsorbed to aluminum hydroxide adjuvant Mildred Ugozzoli a,⇑,1, Donatello Laera b,1, Sandra Nuti b, David A.G. Skibinski b, Simone Bufali b, Chiara Sammicheli b, Simona Tavarini b, Manmohan Singh a, Derek T. O’Hagan a a b
Novartis Vaccines and Diagnostics, Cambridge, MA 02139, USA Novartis Vaccines and Diagnostics, Siena 53100, Italy
a r t i c l e
i n f o
Article history: Received 5 May 2011 Received in revised form 29 June 2011 Accepted 12 July 2011 Available online 22 July 2011 Keywords: Flow cytometry Aluminum hydroxide Vaccine Direct antigen detection Fluorescence Antigen distribution
a b s t r a c t Flow cytometry (FC) has been widely used in biological research; however, its use for vaccine characterization has been very limited. Here we describe the development of an FC method for the direct quantification of two Neisseria meningitidis vaccine antigens, in mono- and multivalent formulations, while still adsorbed on aluminum hydroxide (AH) suspension. The antibody-based method is specific and sensitive. Because FC allows microscopic particle examination, the entire aluminum suspension carrying adsorbed antigen(s) can be analyzed directly. In addition to determining antigen concentration and identity, the assay is able to determine the distribution of the antigens on AH. High correlation coefficients (r2) were routinely achieved for a broad range of antigen doses from 0 to 150 lg/dose. Traditional assays for quantitative and qualitative antigen characterization on AH particles involve either complete aluminum dissolution or antigen desorption from the adjuvant. Because our direct method uses the whole AH suspension, the cumbersome steps used by traditional methods are not required. Those steps are often inefficient in desorbing the antigens and in some cases can lead to protein denaturation. We believe that this novel FC-based assay could circumvent some of the complex and tedious antigen–adjuvant desorption methods. Ó 2011 Elsevier Inc. All rights reserved.
Aluminum-containing adjuvants have been used in vaccine formulation since 1926 to enhance the potency of vaccines. Although this approach has been in use for more than 80 years, not much has changed with regard to understanding the properties and interactions of antigens on aluminum suspensions. Adsorption of antigens to aluminum mineral salts is important to engender potent immunogenic responses. These adsorbed formulations also maximize antigen exposure to the immune system by retaining it at the site of injection [1–3] and aiding uptake into antigen-presenting cells [4–6]. Furthermore, it has been shown that adsorption can stabilize antigens and overcome stability issues observed for various soluble antigens alone [7]. However, the beneficial effect of aluminum adsorption on stabilization is still controversial because conformational changes may alter the thermal resistance of protein antigens. The question remains whether some degree of destabilization induces better or worse immunogenicity [8–11]. Adsorption of the antigen onto the surface of aluminum, therefore, is still considered as important for optimal function of these adjuvants [12],
⇑ Corresponding author. 1
E-mail address: [email protected] (M. Ugozzoli). These authors contributed equally to this work.
0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.07.012
with regulatory requirements outlining that antigens must be adsorbed to aluminum-containing adjuvants. The degree and efficiency of adsorption is most commonly measured by centrifuging the adjuvanted vaccine and assaying the supernatant for unabsorbed antigens by chromatography, colorimetric protein assays, and gel electrophoresis. To establish the identity of the adsorbed antigen, desorption from the adjuvant can be achieved by altering the pH to a point that does not support adsorption or add phosphate, salt, or other agents such as guanidine hydrochloride (GnHCl)2 [13,14]. Dissolution of aluminum hydroxide (AH) and aluminum phosphate with incubation at 37 °C with a 5–10% sodium citrate solution can also be performed to release adsorbed antigen(s) [15]. However, these techniques are often not successful in completely recovering adsorbed proteins, or they can alter the structure of the desorbed proteins. Furthermore, studies looking 2 Abbreviations used: GnHCl, guanidine hydrochloride; AH, aluminum hydroxide; BSA, bovine serum albumin; FC, flow cytometry; GFP, green fluorescent protein; MenB, meningococcal serogroup B; NadA, neisserial adhesion A; NHBA, neisserial heparin-binding antigen; pI, isoelectric point; PBS, phosphate-buffered saline; RT, room temperature; TCA, trichloroacetic acid; APC, allophycocyanin; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; FSC, forward scatter; SSC, side scatter; RFI, relative fluorescence intensity; CV, coefficient of variation.
FC method for direct quantification of antigens / M. Ugozzoli et al. / Anal. Biochem. 418 (2011) 224–230
at the effect of adsorption on antigen structures have found changes in the tertiary structure and thermal stability of model antigens following the adsorption to aluminum adjuvants [9]. Despite these issues, few methods have been reported in the literature describing the direct determination of adsorbed protein concentration, and no method is widely used in vaccine development and manufacturing. It would be of great value to evaluate the structural and chemical stability of the adsorbed antigens on AH without the need for desorption. Direct in situ quantification of antigen while still adsorbed to aluminum adjuvant using enzyme-linked immunosorbent assay (ELISA) has been described [16]. More recently, nearinfrared spectroscopy was used to measure the concentration of bovine serum albumin (BSA) adsorbed onto AH. This nondestructive method is able to directly measure the adsorbed protein concentration in suspension over the linear range of 0.1–1.7 lg/dose [17]. Another recent study described a direct alhydrogel formulation immunoassay (DAFIA) for the quantification of aluminumbound malaria antigen AMA1-C1 over a linear detection range of 0.1–10 lg/ml [18]. Furthermore, the same laboratory developed a generic method for direct quantification of protein adsorbed to aluminum formulations [19]. Here we demonstrate the possibility to use flow cytometry (FC) technology for examining microparticle-based vaccine formulations, in particular, for the detection of antigenic proteins adsorbed on the surface of AH suspensions. FC is a unique technique that allows the analysis of multiple physical characteristics of single particles, usually cells, within heterogeneous populations as they flow into a fluid stream through a beam of light. FC measures the particle’s relative size, relative granularity, and relative fluorescence intensity. It has been heavily used in many traditional applications that are continuously evolving, including protein expression, green fluorescent protein (GFP) transfection [20], cell counting, and immunophenotyping. Lately, FC has expanded into newer applications that enable work on RNA–protein interaction analysis, cell cycle and DNA analysis, cell counting, and apoptosis [21]. To prove this concept, in this article we show the application of FC for the direct detection of two Neisseria meningitidis serogroup B antigens, which are two candidates for a vaccine under clinical evaluation, while still adsorbed on AH. The assay uses as a first step a polyclonal antigen-specific antibody, followed by a secondary antibody conjugated to a fluorophore, whose fluorescence detected by the FC analysis is proportional to the amount of antigen bound to the AH particles. This is in contrast to other methods, such as spectrophotometry and fluorometry, in which the adsorption or transmission percentage of a specific wavelength of light is measured for the entire volume of a sample without discriminating whether the signal is coming from the free antigen or the one bound to the aluminum suspension. Therefore, this FC assay could determine the identity of the adsorbed antigens as well as measure the protein concentration in a single- or multi-antigen-based vaccine. We believe that our method has the potential to find wider application as a preformulation analysis tool in research and development laboratories dedicated to the development of aluminumbased vaccines.
Materials and methods Antigens Clinical-grade material of recombinant proteins for the meningococcal serogroup B (MenB) vaccine was obtained from Novartis Vaccines and Diagnostics (Siena, Italy). The antigen neisserial adhesion A (NadA) [22,23] and the fusion proteins, the neisserial heparin-binding antigen (NHBA) [24,25] fused with GNA1030,
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were used for the study. The theoretical molecular weights confirmed by experimental mass spectrometry were 34,557 kDa for NadA and 67,963 kDa for NHBA–GNA1030, whereas isoelectric points (pI) were theoretically calculated as 4.6 and 5.1, respectively. Antibodies for FC assay Rabbit antisera against NadA and NHBA–GNA1030 were generated by immunization of rabbits with purified recombinant proteins and formulated in AH adjuvant at Novartis Vaccines and Diagnostics (Siena, Italy). The specific polyclonal rabbit antibodies were used at a 1:1000 dilution in 10 mM histidine buffer (pH 6.5). As the final step of the assay, an Alexa Fluor 647 F(ab)2 fragment of goat anti-rabbit antibody purchased from Molecular Probes (Invitrogen) was used at a 1:1600 dilution in 1 phosphate-buffered saline (PBS). Preparation of standards and check-standards AH suspension used for this study was produced at Novartis Vaccines and Diagnostics (Marburg, Germany) and used in licensed vaccines. A set of single-antigen standard suspensions containing NadA or NHBA–GNA1030 at 4.7, 9.4, 18.7, 37.5, 75, and 150 lg/ dose was prepared by serially diluting a stock suspension sample the day before the assay. The stock suspension was prepared by mixing a single dose of AH (1500 lg) with 150 lg of the antigen in 10 mM histidine buffer (pH 6.5). For the detection of NadA and NHBA–GNA1030 in the multivalent vaccine, the standard suspensions were generated by the same procedure as the single antigen; the only modification was that the stock suspension was prepared by mixing 150 lg of each antigen. The final osmolarity of the formulations was adjusted to 300 mOsm/L using a 2-M NaCl solution. The stock suspensions were stirred at 4 °C for a minimum of 2 h, ensuring a complete antigen adsorption. Subsequently, the stock suspensions were serially diluted down to 4.7 lg/dose by mixing at each dilution step 1 ml of AH–antigen suspension with 1 ml of AH suspension, which had been previously adjusted for concentration, pH, and osmolarity. The samples were used in the FC assay after an overnight incubation under a rocking motion at 4 °C. For each assay, blank samples containing 1500 lg/dose of AH, adjusted to pH and osmolarity, were included as part of the standard set. In parallel, six check-standard samples contained 3.1, 6.2, 12.5, 25, 50, and 100 lg/dose, respectively, of each single antigen for the monovalent vaccine; for the multivalent vaccine, both antigens were prepared independently by an individual dilution from another stock suspension sample of 1500 lg/dose of AH with 150 lg/dose of each antigen, exactly as described previously. The dose volume and the amount of AH for each standard and check-standard sample were 500 ll and 1500 lg/dose (human dose), respectively. SDS–PAGE and immunoblotting The aluminum-binding capacity (1500 lg/dose in 10 mM histidine [pH 6.5], 300 mOsm/L) was evaluated for the NadA and NHBA–GNA1030 formulations at 25, 50, 100, 200, 400, and 800 lg/dose in the monovalent vaccine and at 25, 50, 100, 150, and 200 lg/dose in the multivalent vaccine formulations. To determine antigen adsorption, each formulation was centrifuged at 3000g for 10 min and the supernatant was removed without disturbing the pellet. To precipitate the possible unbound antigen, the supernatants were treated with 0.5% deoxycholate sodium salt and incubated for 10 min at room temperature (RT), followed by the addition of 60% trichloroacetic acid (TCA). The TCA pellets were resuspended with sample buffer (Invitrogen), whereas the
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aluminum pellets were reconstituted with sample buffer containing 0.5 M sodium phosphate at pH 8.0. The TCA-treated samples, aluminum pellets, and controls (antigens at 1, 0.5, and 0.25 lg) were loaded into a NUPAGE Novex 4–12% gradient Bis–Tris Midi gel (Invitrogen) and run under reducing conditions at a constant voltage of 200 V for approximately 50 min. Subsequently, the samples were transferred to a nitrocellulose membrane using an iBlot Gel Transfer Device (Invitrogen). After transferring, the membrane was blocked with blocking buffer (5% milk powder and 0.1% Tween 20 in 1 PBS) for 1 h at RT, followed by an overnight incubation at 4 °C with primary polyclonal rabbit against NadA and NHBA– GNA1030 diluted 1:5000 in blocking buffer. After three washes with blocking buffer, the membrane was incubated for 2 h at RT with an anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)-linked antibody from a donkey (Amersham ECL), diluted 1:10,000 in blocking buffer. Following further washes, the blot was developed by incubation with Opti-4CN substrate (BioRad), and the reaction was stopped by washing with MilliQ water. FC assay The antigen NadA was used to set up the assay, and the same conditions were applied for detecting the NHBA–GNA1030 in the monovalent vaccine and both antigens in the multivalent vaccine. The assay consisted of a three-step procedure: saturation of the free aluminum sites with BSA, primary binding with the antibodies that specifically recognize each antigen, and staining with a fluorescence-labeled secondary antibody that reacts with the first antibody. BSA saturation and primary antibody steps require 30 min of incubation at 4 °C using a horizontal gentle agitator, whereas the secondary antibody was incubated for 20 min in the dark at RT without mixing. The blank, standard, and check-standard samples were placed on U96-microwell polypropylene plates (Corning). The 75 ll/well aluminum suspensions were saturated with 25 ll of 10% BSA in 10 mM histidine buffer (pH 6.5). After the incubation, plates were centrifuged (3000g for 5 min) and the supernatants were removed. The aluminum pellets were then washed–centrifuged twice with 100 ll of 10 mM histidine buffer (pH 6.5). To reduce the background, the primary antibodies were preadsorbed with goat serum (Gibco, diluted 1:10 in histidine buffer, pH 6.5) and subsequently diluted down to 1:500 in histidine buffer (pH 6.5). The washed pellets were resuspended with 75 ll of histidine buffer (pH 6.5), and 75 ll of primary antibody was subsequently added. Then the samples were incubated and washed–centrifuged–resuspended as described previously. Goat anti-rabbit antibody Alexa Fluor 647, fluorescently labeled, was diluted 1:800 in 1 PBS and then added to each well. After the incubation, plates were washed–centrifuged and resuspended in 1 PBS. The treated samples were read and acquired on a FACS Canto II (BD Biosciences) using a filter for the allophycocyanin (APC) channel (660/ 20 nm). An additional control included the saturated test sample (AH/NadA) treated with irrelevant primary rabbit antibody followed by the conjugated secondary antibody. Factorial in vitro displacement study An in vitro displacement study was performed to determine whether the protein BSA, used as the blocking agent, or the primary or secondary antibodies interfered in the adsorption of the antigen under evaluation on AH during the assay. The experiment was performed using U96-microwell polypropylene plates. Aliquots of 75 ll of a clinical dose (50 lg/dose) were mixed with the BSA blocking solution, followed by the primary and secondary antibodies. The addition of each reagent was followed by 30 min of incubation at 4 °C, and 20 min for the secondary antibody, followed by washing steps. After each incubation step, all of the superna-
tants were kept for further analysis. The presence of the MenB proteins in the supernatants was evaluated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and the proteins were evidenced by Western blotting using the same polyclonal antibody used for the assay. Results Adsorption of NadA and NHBA–GNA1030 antigens The adsorptive capacity of AH, 1500 lg/dose of adjuvant, was determined after centrifuging the AH suspension and assaying the supernatant by Western blotting for unabsorbed antigens of the monovalent or multivalent vaccines. For the single-antigen vaccines the NadA or NHBA–GNA1030 was adsorbed from 0 to 800 lg/dose, whereas for the multivalent vaccine an equal amount of each antigen from 0 to 200 lg/dose was combined prior to the adsorption. At the clinical antigen dose (50 lg/dose) for the monovalent vaccine, NadA and NHBA–GNA1030 were adsorbed to the AH adjuvant with levels of unabsorbed antigens in the formulation supernatants below the limit of detection (<1%) (data not shown). Furthermore, unabsorbed NadA and NHBA–GNA1030 were not detectable at an antigen concentration up to 200 lg/dose of formulation supernatants, indicating P99% adsorption. Both antigens were instead detectable in monovalent vaccines at 400 lg/dose, indicating that the adsorptive capacity lay between 200 and 400 lg/dose and that at the clinical dose of 50 lg we were significantly distant from the adjuvants’ adsorptive capacity limit of 1500 lg/dose in 10 mM histidine (pH 6.5) (data not shown). However, in the multivalent vaccine, NadA (Fig. 1A) and NHBA– GNA1030 (Fig. 1B) were minimally detected (<1% of the dose) in the supernatants at 100 lg/dose, as determined by comparing the intensity of the control bands after scanning the blots with a scanner–imaging densitometer, indicating that the adsorptive capacity lay between 50 and 100 lg/dose. Establishment of FC assay Autofluorescence of assay components To ensure inherent fluorescence of any components other than the secondary antibody used for staining, we assayed the components for autofluorescence. Analysis of AH by FC identifies 99% of the aggregates having a fluorescence intensity less than 1000 (Fig. 2A). Similarly, the analysis of AH together with assay components, using either mono- or multiantigens, showed no inherent fluorescence over the background level for each antigen (Fig. 2B, NadA) plus the presence of BSA (Fig. 2C) and the primary antibodies (Fig. 2D, anti-NadA). Effect of buffer systems on antigen adsorption Adsorption of antigens to AH adjuvants depends on pH, ionic strength, and the presence of anions such as phosphate [26]. Therefore, it was necessary to evaluate the influence of the assay conditions on the adsorption of the antigens. The amount of antigens that is released from AH during each of the blocking, staining, and washing steps was measured by Western blotting analysis in the supernatant of the assay sample after each of these steps (data not shown). Eluted antigens were not detectable in the supernatants until the incubation with the secondary antibody (diluted in PBS) and the final washing step with PBS. Substitution of PBS with water or 10 mM histidine buffer (pH 7.0) for these final steps was able to alleviate this effect with no detectable antigens released into the supernatants (data not shown). However, because PBS represents the principal component of the fluidic buffer used in the FC system, we decided to maintain
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Fig.1. Western blot evaluation of the adsorption capacity of AH for antigens NadA (A) and NHBA–GNA1030 (B) in multivalent vaccine. The presence of NadA and NHBA– GNA1030 in the supernatant (SN) and pellet (P) phases of the vaccine formulation following centrifugation was determined for multivalent vaccine formulations containing increasing concentrations of both NadA and NHBA–GNA1030 from 0 to 200 lg/dose. The detection of both proteins in the SN at 100 lg/dose indicates that the adsorption capacity of 1500 lg/dose of AH has been exceeded and that the adsorption capacity for AH lay between 50 and 100 lg/dose of both proteins. Western blot was prepared as described in Materials and Methods. Different amounts of antigen (1, 0.5, and 0.25 lg) were added as controls to determine the specificity and sensitivity of the assay. The molecular weights of the protein standards are indicated.
Fig.2. FC determination of background autofluorescence from assay components: AH, NadA, BSA, and rabbit polyclonal antibody specific for NadA. Histograms represent events counted versus fluorescence intensity detected for the APC channel 660/20. Analysis of AH alone by FC identified 99% of the particles having fluorescence intensity less than 1000 (A). Similar analysis of AH together with the assay components indicated no inherent fluorescence over the background level for the antigen NadA, for the blocking agent BSA, or by the primary antibody (B–D). RFI, relative fluorescence intensity.
the final steps in PBS to avoid problems of equilibration when the samples entered the FC. Furthermore, despite these observations, the antigens remained predominantly bound to AH with the recovery of the majority of the antigens from AH once the antibodystaining procedure had been completed. Specificity of antibody binding against aluminum-adsorbed antigens The analysis of the standard formulations’ fluorescence intensity confirmed that for AH at 1500 lg/dose in the absence
of antigens, most of the adjuvant aggregates (>99%) had a fluorescence intensity less than 1000. However, for the diluted sample containing NadA at a concentration of 50 lg/dose (human dose), the aggregates showed a marked increase in relative fluorescence with a mean relative fluorescence 9.6-fold greater than that observed for the negative control (0 lg/dose) formulation (Fig. 3A). Because antibodies have been reported to adsorb to the AH adjuvant via a ligand exchange mechanism and electrostatic
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Fig.3. Specificity of antibody binding against aluminum-adsorbed antigen. The histograms represent the events counted as percentage (%) versus the relative fluorescence intensity measured for the APC–Alexa Fluor 647 at channel 660/20 for various sample controls. (A) FC assay of AH formulations with 0 and 50 lg/dose of NadA (red and blue lines, respectively). (B) Nonspecific binding of the secondary antibody was determined by performing the FC assay in the absence of NadA with primary antibody diluted 1:1000 and 1:10,000 (red and blue lines, respectively) or containing 50 lg/dose of NadA but using irrelevant primary antibody (green line). (C) Reproducibility of the assay was determined for NadA analyzing identical formulations 24 h apart (red and green lines) or by waiting 24 h following the antibody-staining procedure before FC analysis (blue line).
interactions [27], it was necessary to investigate whether any unspecific binding of the primary and secondary antibodies to AH could lead to artificial fluorescence readouts. As expected, not only was a high fluorescence value measured for the negative control, but also similar fluorescence intensity measurements were detected for the monovalent vaccine containing NadA from 25 to 800 lg/dose (data not shown). Therefore, to counter the nonspecific binding, AH was blocked with 2.5% BSA prior to staining with the primary and secondary antibodies. This blocking was successful in limiting nonspecific binding given that performing the FC assay in the absence of antigen with primary antibodies diluted 1:1000 or 1:10,000 (Fig. 3B) resulted in levels of fluorescence for the AH particles 10-fold below levels observed for the complete assay with NadA formulated at 50 lg/dose (Fig. 3A). This result confirmed the essential role of BSA treatment as the blocking agent in the development of this assay. Furthermore, when the assay was performed with irrelevant primary antibodies not specific for the protein under assessment, the levels of fluorescence remained equal to those observed in the absence of antigen, indicating that nonspecific binding between the antigen and the fluorescently labeled secondary antibody was not significant (Fig. 3B). To evaluate the stability and reproducibility of the assay, multiple formulations were prepared in parallel but stained and/or analyzed by FC at different time intervals (Fig. 3C, NadA). The assay is highly reproducible given that little variation was observed between identical formulations assayed 24 h apart. Furthermore, the assay is stable given that waiting 24 h following the antibody staining procedure had little impact on the levels of fluorescence measured by FC analysis.
Morphology and antigen distribution of standard formulations Standard formulations, prepared from a stock solution (150 lg/ dose of NadA) and serially diluted with aluminum alone (down to 4.7 lg/dose), were analyzed by FC using both forward scatter (FSC) and side scatter (SSC) parameters, which are associated with particle size and morphology, respectively. All standard formulations, for both mono- and multivalent vaccines, appeared as a single uniform population of particles (Fig. 4A, NadA), with increased concentration of adsorbed antigen having no impact on FSC and SSC measurements. Antigen distribution on AH aggregates was analyzed using SSC and relative fluorescence intensity (RFI). The analysis of the RFI revealed that when the suspension had no antigen adsorbed, 99% of the aggregates showed an RFI <1000. However, as the concentration of the antigens gradually increased, the mean fluorescence intensity increased proportionally with the amount of antigen adsorbed (Fig. 4B, NadA). When AH particles were loaded with the highest concentration of NadA (150 lg/dose), the entire
suspension exposed a uniformly distributed fluorescence 23-fold higher than AH alone. Direct quantification of adsorbed antigens in vaccine formulations Standard formulations were prepared in the range of 0–150 lg/ dose and tested by the FC assay. Standard curves generated by three-parameter nonlinear regression analysis had correlation coefficients R2 > 0.99 (Fig. 5). To evaluate the reliability of the standard curves in measuring the antigen concentrations, a series of check-standard samples ranging from 3 to 100 lg/dose were assayed. The protein concentrations were determined from fluorescence readings using the y = ax3 + bx2 + cx regression equation (Table 1). The antigen concentrations calculated were in agreement with the nominal amount for both of the antigens, NadA and NHBA–GNA1030, with the overall percentage of accuracies being 85–97% for the detection range of 3.1–100 lg/dose in the monovalent vaccines and 80–97% for the detection range of 3.1–50 lg/dose in the multivalent vaccine. Interassay variation analysis showed that the coefficients of variation (CVs) of test samples for the range from 3.1 to 50 lg/dose were consistently low. The CV results obtained from triplicate samples performed in three independent assays were shown to be acceptable from 1% to 6%, indicating that the assay is highly reproducible within the described range for antigens in both the mono- and multivalent vaccines. Discussion Traditional methods for determining the protein antigen concentration of aluminum-based vaccines require the removal of the antigen from the adjuvant surface, a process that is often time-consuming. These methods expose the problems of either incomplete or partial recovery of the adsorbed proteins and modification of the protein structure during the desorption process. For these reasons, the degree of antigen adsorption to aluminumbased adjuvants is most commonly measured by centrifuging the adsorbed vaccine and assaying the supernatant for unabsorbed antigens by chromatography, colorimetric protein assays, and gel electrophoresis. A number of recent studies [17–19] have reported the direct in situ quantification of antigens; however, further exploration in this area is required before this approach can be accepted and widely used in vaccine development and manufacturing. Here we have demonstrated the usefulness of a technique that has long been used in the cell biology field as a novel application, that is, the quality control assessment of adjuvanted formulations by FC. The great advantage of the FC technology is the possibility to use the whole formulation for direct qualitative and quantitative
FC method for direct quantification of antigens / M. Ugozzoli et al. / Anal. Biochem. 418 (2011) 224–230
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Fig.4. Morphology and antigen distribution for standard formulations. (A) Representative two-dimensional dot plot indicating common morphological distribution of AH particles observed for all standard formulations (0–150 lg/dose). The gated dots appeared as a single uniform population of particles, with increased concentration of adsorbed antigens having no impact on FSC (x axis) and SSC (y axis) measurements. (B) Analysis of antigen distribution of NadA on AH aggregates using SSC and relative fluorescence intensity (RFI). The concentration of NadA in the formulation is indicated for each individual plot. The percentage of fluorescent particles increases with the concentration of NadA (0–150 lg/dose).
Fig.5. Representative standard curve for FC assay to determine antigen concentration of AH formulations. A standard curve was generated using a three-parameter nonlinear regression analysis, y = ax3 + bx2 + cx, with correlation coefficients R2 > 0.99. The y axis displays the percentage of particles with RFI > 1000. Data are the means of triplicate samples performed on three independent experiments. Error bars show standard deviations between the assays.
analysis, especially on those formulations that are based on microparticle emulsions [28] or suspensions such as the aluminumbased formulations, representing the most common platform used in licensed vaccines. The method we developed is highly accurate in determining the concentration of adsorbed proteins (85–97% in monovalent formulations, 80–97% in multivalent formulations) within a broad detection range of 3.1–100 lg/dose, with sufficient
sensitivity to detect antigen concentrations as low as 3.1 lg/dose. Furthermore, we showed that the assay is reproducible and stable, with little variations observed between formulations assayed at different times or with a time delay between the staining procedure and the FC analysis of the samples. In addition to the accuracy, sensitivity, and reproducibility, we believe that the FC assay possesses a number of other key advantages that originate from the fact of analyzing physical particles; FC detects the antigen directly on the surface of the aluminum particles. First, the degree of antigen adsorption to aluminum is measured directly without the need to rely on indirect measurements of unadsorbed antigen in the formulation of supernatants. Second, because the assay is antibody based, it is possible to use FC to monitor antigen stability while still bound to the surface of aluminumcontaining adjuvants. This could be particularly powerful for antigens where protective epitopes have been well defined and where monoclonal antibodies, specific for these regions, could be used in the FC assay to monitor the structural integrity of the epitopes over time or to monitor vaccine consistency prior to their release. Notably, a clear advantage of this assay is the ability to determine protein concentrations on each aluminum adjuvant particle, giving insight to the antigen distribution within the formulation. With guidelines in the literature [29] describing different adsorption strategies for the production of multicomponent vaccines, FCbased assays could help to monitor the uniformity of resulting vaccines and the importance of antigen distribution for efficacy. Finally, another common advantage for antibody-based assays specific for a target antigen is the potential of the assay to be
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Table 1 Results from check-standard samples for NadA (A) and NHBA-GNA1030 (B) in monovalent vaccine and for NadA (C) and NHBA-GNA1030 (D) in multivalent vaccine. Nominal concentration (lg)
Concentration detected (lg)
Accuracy (%)
(A) NadA concentrations of monovalent formulated vaccine 3.1 3.0 ± 0.1 94.8 ± 2.1 6.2 6.3 ± 0.3 96.4 ± 2.8 12.5 12.8 ± 0.2 97.6 ± 1.6 25.0 24.9 ± 0.8 97.5 ± 2.0 50.0 50.5 ± 2.8 94.8 ± 1.5 100.0 113.9 ± 12.3 84.7 ± 10.1
CV (%) 4.8 4.8 1.6 3.4 5.7 10.8
(B) NHBA-GNA1030 concentrations of monovalent formulated vaccine 3.12 2.99 ± 0.1 95.94 ± 3.92 6.25 5.69 ± 0.3 91.09 ± 5.52 12.5 11.26 ± 0.4 90.11 ± 2.86 25 23.53 ± 0.4 94.13 ± 1.66 50 46.57 ± 0.9 93.15 ± 1.87 100 107.57 ± 1.1 92.43 ± 1.11
4.08 6.07 3.17 1.76 2.00 1.03
(C) NadA concentrations of multivalent formulated vaccine 3.12 3.07 ± 0.11 97.22 ± 2.06 6.25 6.43 ± 0.13 97.17 ± 2.03 12.5 13.11 ± 0.6 95.09 ± 4.81 25 23.31 ± 0.55 93.25 ± 2.19 50 40.59 ± 1.7 81.19 ± 3.39 100 135.61 ± 24.61 64.39 ± 24.61
3.58 1.97 4.59 2.35 4.18 18.15
(D) NHBA-GNA1030 concentrations of multivalent formulated vaccine 3.12 2.99 ± 0.18 95.94 ± 5.7 6.25 6.16 ± 0.4 95.57 ± 3.76 12.5 12.16 ± 0.24 97.31 ± 1.9 25 22.65 ± 0.55 90.61 ± 2.21 50 40.16 ± 2.04 80.31 ± 4.08 100 127.61 ± 8.74 72.39 ± 8.74
[5]
[6]
[7]
[8] [9]
[10]
[11]
[12]
[13]
[14] 5.94 6.45 1.95 2.44 5.08 6.85
Note: All of the results were obtained from triplicate samples representing three independent experiments. Check-standard samples of antigens formulated with AH were freshly prepared and diluted to a final concentration with AH suspension (1500 lg/dose). Back calculations were performed by converting the percentage of fluorescent particles measured for the check-standard concentrations of antigen using three-parameter nonlinear regression analysis. % Accuracy is the percentage of similarity between the amount of antigens calculated by back calculation (detected by FC assay) and the known amount of antigens. % CV is the coefficient of variation between assays and is calculated as the standard deviation over the mean.
applied directly to complex formulations containing multiple antigens and adjuvants such as combination vaccines. To conclude, we have demonstrated that FC is a powerful tool for the characterization of AH-based vaccine formulations. Our FC-based assay is antigen specific, sensitive, and reproducible. The assay has several other potential applications, including monitoring long-term antigen stability, distribution without antigen desorption, and the possibility to extend the analysis to other adjuvants and delivery systems such as emulsions, polymeric microparticles, and liposomes.
[15]
[16]
[17]
[18]
[19]
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[21] [22]
[23]
[24]
Acknowledgments The authors thank Rino Rappuoli, global head (research) of Novartis Vaccines and Diagnostics, for support in this work. The authors also acknowledge the support provided by Michele Pallaoro.
[25]
[26]
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Stroop, Physiochemical and functional characterization of antigen proteins eluted from aluminum hydroxide adjuvant, Vaccine 24 (2006) 7214–7225. S.J. Seeber, J.L. White, S.L. Hem, Solubilization of aluminum-containing adjuvants by constituents of interstitial fluid, J. Parenteral. Sci. Technol. 45 (1991) 156–159. J.B. Katz, S.K. Hanson, P.A. Patterson, I.R. Stoll, In vitro assessment of viral antigen content in inactivated aluminum hydroxide adjuvanted vaccines, J. Virol. Methods 25 (1989) 101–108. X.X. Lai, Y.W. Zheng, S. Jacobsen, J.N. Larsen, H. Ipsen, H. Lowenstein, I. Sondergaard, Determination of adsorbed protein concentration in aluminum hydroxide suspensions by near-infrared transmittance spectroscopy, Appl. Spectrosc. 62 (2008) 784–790. D.M. Zhu, S.H. Huang, E. Gebregeorgis, H. McClellan, W.L. Dai, L. Miller, A. Sau, Development of a direct alhydrogel formulation immunoassay (DAFIA), J. Immunol. Methods 344 (2009) 73–78. D.M. Zhu, A. Saul, S.H. Huang, L.B. Martin, L.H. 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Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Flow cytometry: An alternative method for direct quantification of antigens adsorbed to aluminum hydroxide adjuvant Mildred Ugozzoli a,⇑,1, Donatello Laera b,1, Sandra Nuti b, David A.G. Skibinski b, Simone Bufali b, Chiara Sammicheli b, Simona Tavarini b, Manmohan Singh a, Derek T. O’Hagan a a b
Novartis Vaccines and Diagnostics, Cambridge, MA 02139, USA Novartis Vaccines and Diagnostics, Siena 53100, Italy
a r t i c l e
i n f o
Article history: Received 5 May 2011 Received in revised form 29 June 2011 Accepted 12 July 2011 Available online 22 July 2011 Keywords: Flow cytometry Aluminum hydroxide Vaccine Direct antigen detection Fluorescence Antigen distribution
a b s t r a c t Flow cytometry (FC) has been widely used in biological research; however, its use for vaccine characterization has been very limited. Here we describe the development of an FC method for the direct quantification of two Neisseria meningitidis vaccine antigens, in mono- and multivalent formulations, while still adsorbed on aluminum hydroxide (AH) suspension. The antibody-based method is specific and sensitive. Because FC allows microscopic particle examination, the entire aluminum suspension carrying adsorbed antigen(s) can be analyzed directly. In addition to determining antigen concentration and identity, the assay is able to determine the distribution of the antigens on AH. High correlation coefficients (r2) were routinely achieved for a broad range of antigen doses from 0 to 150 lg/dose. Traditional assays for quantitative and qualitative antigen characterization on AH particles involve either complete aluminum dissolution or antigen desorption from the adjuvant. Because our direct method uses the whole AH suspension, the cumbersome steps used by traditional methods are not required. Those steps are often inefficient in desorbing the antigens and in some cases can lead to protein denaturation. We believe that this novel FC-based assay could circumvent some of the complex and tedious antigen–adjuvant desorption methods. Ó 2011 Elsevier Inc. All rights reserved.
Aluminum-containing adjuvants have been used in vaccine formulation since 1926 to enhance the potency of vaccines. Although this approach has been in use for more than 80 years, not much has changed with regard to understanding the properties and interactions of antigens on aluminum suspensions. Adsorption of antigens to aluminum mineral salts is important to engender potent immunogenic responses. These adsorbed formulations also maximize antigen exposure to the immune system by retaining it at the site of injection [1–3] and aiding uptake into antigen-presenting cells [4–6]. Furthermore, it has been shown that adsorption can stabilize antigens and overcome stability issues observed for various soluble antigens alone [7]. However, the beneficial effect of aluminum adsorption on stabilization is still controversial because conformational changes may alter the thermal resistance of protein antigens. The question remains whether some degree of destabilization induces better or worse immunogenicity [8–11]. Adsorption of the antigen onto the surface of aluminum, therefore, is still considered as important for optimal function of these adjuvants [12],
⇑ Corresponding author. 1
E-mail address: [email protected] (M. Ugozzoli). These authors contributed equally to this work.
0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.07.012
with regulatory requirements outlining that antigens must be adsorbed to aluminum-containing adjuvants. The degree and efficiency of adsorption is most commonly measured by centrifuging the adjuvanted vaccine and assaying the supernatant for unabsorbed antigens by chromatography, colorimetric protein assays, and gel electrophoresis. To establish the identity of the adsorbed antigen, desorption from the adjuvant can be achieved by altering the pH to a point that does not support adsorption or add phosphate, salt, or other agents such as guanidine hydrochloride (GnHCl)2 [13,14]. Dissolution of aluminum hydroxide (AH) and aluminum phosphate with incubation at 37 °C with a 5–10% sodium citrate solution can also be performed to release adsorbed antigen(s) [15]. However, these techniques are often not successful in completely recovering adsorbed proteins, or they can alter the structure of the desorbed proteins. Furthermore, studies looking 2 Abbreviations used: GnHCl, guanidine hydrochloride; AH, aluminum hydroxide; BSA, bovine serum albumin; FC, flow cytometry; GFP, green fluorescent protein; MenB, meningococcal serogroup B; NadA, neisserial adhesion A; NHBA, neisserial heparin-binding antigen; pI, isoelectric point; PBS, phosphate-buffered saline; RT, room temperature; TCA, trichloroacetic acid; APC, allophycocyanin; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; FSC, forward scatter; SSC, side scatter; RFI, relative fluorescence intensity; CV, coefficient of variation.
FC method for direct quantification of antigens / M. Ugozzoli et al. / Anal. Biochem. 418 (2011) 224–230
at the effect of adsorption on antigen structures have found changes in the tertiary structure and thermal stability of model antigens following the adsorption to aluminum adjuvants [9]. Despite these issues, few methods have been reported in the literature describing the direct determination of adsorbed protein concentration, and no method is widely used in vaccine development and manufacturing. It would be of great value to evaluate the structural and chemical stability of the adsorbed antigens on AH without the need for desorption. Direct in situ quantification of antigen while still adsorbed to aluminum adjuvant using enzyme-linked immunosorbent assay (ELISA) has been described [16]. More recently, nearinfrared spectroscopy was used to measure the concentration of bovine serum albumin (BSA) adsorbed onto AH. This nondestructive method is able to directly measure the adsorbed protein concentration in suspension over the linear range of 0.1–1.7 lg/dose [17]. Another recent study described a direct alhydrogel formulation immunoassay (DAFIA) for the quantification of aluminumbound malaria antigen AMA1-C1 over a linear detection range of 0.1–10 lg/ml [18]. Furthermore, the same laboratory developed a generic method for direct quantification of protein adsorbed to aluminum formulations [19]. Here we demonstrate the possibility to use flow cytometry (FC) technology for examining microparticle-based vaccine formulations, in particular, for the detection of antigenic proteins adsorbed on the surface of AH suspensions. FC is a unique technique that allows the analysis of multiple physical characteristics of single particles, usually cells, within heterogeneous populations as they flow into a fluid stream through a beam of light. FC measures the particle’s relative size, relative granularity, and relative fluorescence intensity. It has been heavily used in many traditional applications that are continuously evolving, including protein expression, green fluorescent protein (GFP) transfection [20], cell counting, and immunophenotyping. Lately, FC has expanded into newer applications that enable work on RNA–protein interaction analysis, cell cycle and DNA analysis, cell counting, and apoptosis [21]. To prove this concept, in this article we show the application of FC for the direct detection of two Neisseria meningitidis serogroup B antigens, which are two candidates for a vaccine under clinical evaluation, while still adsorbed on AH. The assay uses as a first step a polyclonal antigen-specific antibody, followed by a secondary antibody conjugated to a fluorophore, whose fluorescence detected by the FC analysis is proportional to the amount of antigen bound to the AH particles. This is in contrast to other methods, such as spectrophotometry and fluorometry, in which the adsorption or transmission percentage of a specific wavelength of light is measured for the entire volume of a sample without discriminating whether the signal is coming from the free antigen or the one bound to the aluminum suspension. Therefore, this FC assay could determine the identity of the adsorbed antigens as well as measure the protein concentration in a single- or multi-antigen-based vaccine. We believe that our method has the potential to find wider application as a preformulation analysis tool in research and development laboratories dedicated to the development of aluminumbased vaccines.
Materials and methods Antigens Clinical-grade material of recombinant proteins for the meningococcal serogroup B (MenB) vaccine was obtained from Novartis Vaccines and Diagnostics (Siena, Italy). The antigen neisserial adhesion A (NadA) [22,23] and the fusion proteins, the neisserial heparin-binding antigen (NHBA) [24,25] fused with GNA1030,
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were used for the study. The theoretical molecular weights confirmed by experimental mass spectrometry were 34,557 kDa for NadA and 67,963 kDa for NHBA–GNA1030, whereas isoelectric points (pI) were theoretically calculated as 4.6 and 5.1, respectively. Antibodies for FC assay Rabbit antisera against NadA and NHBA–GNA1030 were generated by immunization of rabbits with purified recombinant proteins and formulated in AH adjuvant at Novartis Vaccines and Diagnostics (Siena, Italy). The specific polyclonal rabbit antibodies were used at a 1:1000 dilution in 10 mM histidine buffer (pH 6.5). As the final step of the assay, an Alexa Fluor 647 F(ab)2 fragment of goat anti-rabbit antibody purchased from Molecular Probes (Invitrogen) was used at a 1:1600 dilution in 1 phosphate-buffered saline (PBS). Preparation of standards and check-standards AH suspension used for this study was produced at Novartis Vaccines and Diagnostics (Marburg, Germany) and used in licensed vaccines. A set of single-antigen standard suspensions containing NadA or NHBA–GNA1030 at 4.7, 9.4, 18.7, 37.5, 75, and 150 lg/ dose was prepared by serially diluting a stock suspension sample the day before the assay. The stock suspension was prepared by mixing a single dose of AH (1500 lg) with 150 lg of the antigen in 10 mM histidine buffer (pH 6.5). For the detection of NadA and NHBA–GNA1030 in the multivalent vaccine, the standard suspensions were generated by the same procedure as the single antigen; the only modification was that the stock suspension was prepared by mixing 150 lg of each antigen. The final osmolarity of the formulations was adjusted to 300 mOsm/L using a 2-M NaCl solution. The stock suspensions were stirred at 4 °C for a minimum of 2 h, ensuring a complete antigen adsorption. Subsequently, the stock suspensions were serially diluted down to 4.7 lg/dose by mixing at each dilution step 1 ml of AH–antigen suspension with 1 ml of AH suspension, which had been previously adjusted for concentration, pH, and osmolarity. The samples were used in the FC assay after an overnight incubation under a rocking motion at 4 °C. For each assay, blank samples containing 1500 lg/dose of AH, adjusted to pH and osmolarity, were included as part of the standard set. In parallel, six check-standard samples contained 3.1, 6.2, 12.5, 25, 50, and 100 lg/dose, respectively, of each single antigen for the monovalent vaccine; for the multivalent vaccine, both antigens were prepared independently by an individual dilution from another stock suspension sample of 1500 lg/dose of AH with 150 lg/dose of each antigen, exactly as described previously. The dose volume and the amount of AH for each standard and check-standard sample were 500 ll and 1500 lg/dose (human dose), respectively. SDS–PAGE and immunoblotting The aluminum-binding capacity (1500 lg/dose in 10 mM histidine [pH 6.5], 300 mOsm/L) was evaluated for the NadA and NHBA–GNA1030 formulations at 25, 50, 100, 200, 400, and 800 lg/dose in the monovalent vaccine and at 25, 50, 100, 150, and 200 lg/dose in the multivalent vaccine formulations. To determine antigen adsorption, each formulation was centrifuged at 3000g for 10 min and the supernatant was removed without disturbing the pellet. To precipitate the possible unbound antigen, the supernatants were treated with 0.5% deoxycholate sodium salt and incubated for 10 min at room temperature (RT), followed by the addition of 60% trichloroacetic acid (TCA). The TCA pellets were resuspended with sample buffer (Invitrogen), whereas the
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aluminum pellets were reconstituted with sample buffer containing 0.5 M sodium phosphate at pH 8.0. The TCA-treated samples, aluminum pellets, and controls (antigens at 1, 0.5, and 0.25 lg) were loaded into a NUPAGE Novex 4–12% gradient Bis–Tris Midi gel (Invitrogen) and run under reducing conditions at a constant voltage of 200 V for approximately 50 min. Subsequently, the samples were transferred to a nitrocellulose membrane using an iBlot Gel Transfer Device (Invitrogen). After transferring, the membrane was blocked with blocking buffer (5% milk powder and 0.1% Tween 20 in 1 PBS) for 1 h at RT, followed by an overnight incubation at 4 °C with primary polyclonal rabbit against NadA and NHBA– GNA1030 diluted 1:5000 in blocking buffer. After three washes with blocking buffer, the membrane was incubated for 2 h at RT with an anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)-linked antibody from a donkey (Amersham ECL), diluted 1:10,000 in blocking buffer. Following further washes, the blot was developed by incubation with Opti-4CN substrate (BioRad), and the reaction was stopped by washing with MilliQ water. FC assay The antigen NadA was used to set up the assay, and the same conditions were applied for detecting the NHBA–GNA1030 in the monovalent vaccine and both antigens in the multivalent vaccine. The assay consisted of a three-step procedure: saturation of the free aluminum sites with BSA, primary binding with the antibodies that specifically recognize each antigen, and staining with a fluorescence-labeled secondary antibody that reacts with the first antibody. BSA saturation and primary antibody steps require 30 min of incubation at 4 °C using a horizontal gentle agitator, whereas the secondary antibody was incubated for 20 min in the dark at RT without mixing. The blank, standard, and check-standard samples were placed on U96-microwell polypropylene plates (Corning). The 75 ll/well aluminum suspensions were saturated with 25 ll of 10% BSA in 10 mM histidine buffer (pH 6.5). After the incubation, plates were centrifuged (3000g for 5 min) and the supernatants were removed. The aluminum pellets were then washed–centrifuged twice with 100 ll of 10 mM histidine buffer (pH 6.5). To reduce the background, the primary antibodies were preadsorbed with goat serum (Gibco, diluted 1:10 in histidine buffer, pH 6.5) and subsequently diluted down to 1:500 in histidine buffer (pH 6.5). The washed pellets were resuspended with 75 ll of histidine buffer (pH 6.5), and 75 ll of primary antibody was subsequently added. Then the samples were incubated and washed–centrifuged–resuspended as described previously. Goat anti-rabbit antibody Alexa Fluor 647, fluorescently labeled, was diluted 1:800 in 1 PBS and then added to each well. After the incubation, plates were washed–centrifuged and resuspended in 1 PBS. The treated samples were read and acquired on a FACS Canto II (BD Biosciences) using a filter for the allophycocyanin (APC) channel (660/ 20 nm). An additional control included the saturated test sample (AH/NadA) treated with irrelevant primary rabbit antibody followed by the conjugated secondary antibody. Factorial in vitro displacement study An in vitro displacement study was performed to determine whether the protein BSA, used as the blocking agent, or the primary or secondary antibodies interfered in the adsorption of the antigen under evaluation on AH during the assay. The experiment was performed using U96-microwell polypropylene plates. Aliquots of 75 ll of a clinical dose (50 lg/dose) were mixed with the BSA blocking solution, followed by the primary and secondary antibodies. The addition of each reagent was followed by 30 min of incubation at 4 °C, and 20 min for the secondary antibody, followed by washing steps. After each incubation step, all of the superna-
tants were kept for further analysis. The presence of the MenB proteins in the supernatants was evaluated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and the proteins were evidenced by Western blotting using the same polyclonal antibody used for the assay. Results Adsorption of NadA and NHBA–GNA1030 antigens The adsorptive capacity of AH, 1500 lg/dose of adjuvant, was determined after centrifuging the AH suspension and assaying the supernatant by Western blotting for unabsorbed antigens of the monovalent or multivalent vaccines. For the single-antigen vaccines the NadA or NHBA–GNA1030 was adsorbed from 0 to 800 lg/dose, whereas for the multivalent vaccine an equal amount of each antigen from 0 to 200 lg/dose was combined prior to the adsorption. At the clinical antigen dose (50 lg/dose) for the monovalent vaccine, NadA and NHBA–GNA1030 were adsorbed to the AH adjuvant with levels of unabsorbed antigens in the formulation supernatants below the limit of detection (<1%) (data not shown). Furthermore, unabsorbed NadA and NHBA–GNA1030 were not detectable at an antigen concentration up to 200 lg/dose of formulation supernatants, indicating P99% adsorption. Both antigens were instead detectable in monovalent vaccines at 400 lg/dose, indicating that the adsorptive capacity lay between 200 and 400 lg/dose and that at the clinical dose of 50 lg we were significantly distant from the adjuvants’ adsorptive capacity limit of 1500 lg/dose in 10 mM histidine (pH 6.5) (data not shown). However, in the multivalent vaccine, NadA (Fig. 1A) and NHBA– GNA1030 (Fig. 1B) were minimally detected (<1% of the dose) in the supernatants at 100 lg/dose, as determined by comparing the intensity of the control bands after scanning the blots with a scanner–imaging densitometer, indicating that the adsorptive capacity lay between 50 and 100 lg/dose. Establishment of FC assay Autofluorescence of assay components To ensure inherent fluorescence of any components other than the secondary antibody used for staining, we assayed the components for autofluorescence. Analysis of AH by FC identifies 99% of the aggregates having a fluorescence intensity less than 1000 (Fig. 2A). Similarly, the analysis of AH together with assay components, using either mono- or multiantigens, showed no inherent fluorescence over the background level for each antigen (Fig. 2B, NadA) plus the presence of BSA (Fig. 2C) and the primary antibodies (Fig. 2D, anti-NadA). Effect of buffer systems on antigen adsorption Adsorption of antigens to AH adjuvants depends on pH, ionic strength, and the presence of anions such as phosphate [26]. Therefore, it was necessary to evaluate the influence of the assay conditions on the adsorption of the antigens. The amount of antigens that is released from AH during each of the blocking, staining, and washing steps was measured by Western blotting analysis in the supernatant of the assay sample after each of these steps (data not shown). Eluted antigens were not detectable in the supernatants until the incubation with the secondary antibody (diluted in PBS) and the final washing step with PBS. Substitution of PBS with water or 10 mM histidine buffer (pH 7.0) for these final steps was able to alleviate this effect with no detectable antigens released into the supernatants (data not shown). However, because PBS represents the principal component of the fluidic buffer used in the FC system, we decided to maintain
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Fig.1. Western blot evaluation of the adsorption capacity of AH for antigens NadA (A) and NHBA–GNA1030 (B) in multivalent vaccine. The presence of NadA and NHBA– GNA1030 in the supernatant (SN) and pellet (P) phases of the vaccine formulation following centrifugation was determined for multivalent vaccine formulations containing increasing concentrations of both NadA and NHBA–GNA1030 from 0 to 200 lg/dose. The detection of both proteins in the SN at 100 lg/dose indicates that the adsorption capacity of 1500 lg/dose of AH has been exceeded and that the adsorption capacity for AH lay between 50 and 100 lg/dose of both proteins. Western blot was prepared as described in Materials and Methods. Different amounts of antigen (1, 0.5, and 0.25 lg) were added as controls to determine the specificity and sensitivity of the assay. The molecular weights of the protein standards are indicated.
Fig.2. FC determination of background autofluorescence from assay components: AH, NadA, BSA, and rabbit polyclonal antibody specific for NadA. Histograms represent events counted versus fluorescence intensity detected for the APC channel 660/20. Analysis of AH alone by FC identified 99% of the particles having fluorescence intensity less than 1000 (A). Similar analysis of AH together with the assay components indicated no inherent fluorescence over the background level for the antigen NadA, for the blocking agent BSA, or by the primary antibody (B–D). RFI, relative fluorescence intensity.
the final steps in PBS to avoid problems of equilibration when the samples entered the FC. Furthermore, despite these observations, the antigens remained predominantly bound to AH with the recovery of the majority of the antigens from AH once the antibodystaining procedure had been completed. Specificity of antibody binding against aluminum-adsorbed antigens The analysis of the standard formulations’ fluorescence intensity confirmed that for AH at 1500 lg/dose in the absence
of antigens, most of the adjuvant aggregates (>99%) had a fluorescence intensity less than 1000. However, for the diluted sample containing NadA at a concentration of 50 lg/dose (human dose), the aggregates showed a marked increase in relative fluorescence with a mean relative fluorescence 9.6-fold greater than that observed for the negative control (0 lg/dose) formulation (Fig. 3A). Because antibodies have been reported to adsorb to the AH adjuvant via a ligand exchange mechanism and electrostatic
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Fig.3. Specificity of antibody binding against aluminum-adsorbed antigen. The histograms represent the events counted as percentage (%) versus the relative fluorescence intensity measured for the APC–Alexa Fluor 647 at channel 660/20 for various sample controls. (A) FC assay of AH formulations with 0 and 50 lg/dose of NadA (red and blue lines, respectively). (B) Nonspecific binding of the secondary antibody was determined by performing the FC assay in the absence of NadA with primary antibody diluted 1:1000 and 1:10,000 (red and blue lines, respectively) or containing 50 lg/dose of NadA but using irrelevant primary antibody (green line). (C) Reproducibility of the assay was determined for NadA analyzing identical formulations 24 h apart (red and green lines) or by waiting 24 h following the antibody-staining procedure before FC analysis (blue line).
interactions [27], it was necessary to investigate whether any unspecific binding of the primary and secondary antibodies to AH could lead to artificial fluorescence readouts. As expected, not only was a high fluorescence value measured for the negative control, but also similar fluorescence intensity measurements were detected for the monovalent vaccine containing NadA from 25 to 800 lg/dose (data not shown). Therefore, to counter the nonspecific binding, AH was blocked with 2.5% BSA prior to staining with the primary and secondary antibodies. This blocking was successful in limiting nonspecific binding given that performing the FC assay in the absence of antigen with primary antibodies diluted 1:1000 or 1:10,000 (Fig. 3B) resulted in levels of fluorescence for the AH particles 10-fold below levels observed for the complete assay with NadA formulated at 50 lg/dose (Fig. 3A). This result confirmed the essential role of BSA treatment as the blocking agent in the development of this assay. Furthermore, when the assay was performed with irrelevant primary antibodies not specific for the protein under assessment, the levels of fluorescence remained equal to those observed in the absence of antigen, indicating that nonspecific binding between the antigen and the fluorescently labeled secondary antibody was not significant (Fig. 3B). To evaluate the stability and reproducibility of the assay, multiple formulations were prepared in parallel but stained and/or analyzed by FC at different time intervals (Fig. 3C, NadA). The assay is highly reproducible given that little variation was observed between identical formulations assayed 24 h apart. Furthermore, the assay is stable given that waiting 24 h following the antibody staining procedure had little impact on the levels of fluorescence measured by FC analysis.
Morphology and antigen distribution of standard formulations Standard formulations, prepared from a stock solution (150 lg/ dose of NadA) and serially diluted with aluminum alone (down to 4.7 lg/dose), were analyzed by FC using both forward scatter (FSC) and side scatter (SSC) parameters, which are associated with particle size and morphology, respectively. All standard formulations, for both mono- and multivalent vaccines, appeared as a single uniform population of particles (Fig. 4A, NadA), with increased concentration of adsorbed antigen having no impact on FSC and SSC measurements. Antigen distribution on AH aggregates was analyzed using SSC and relative fluorescence intensity (RFI). The analysis of the RFI revealed that when the suspension had no antigen adsorbed, 99% of the aggregates showed an RFI <1000. However, as the concentration of the antigens gradually increased, the mean fluorescence intensity increased proportionally with the amount of antigen adsorbed (Fig. 4B, NadA). When AH particles were loaded with the highest concentration of NadA (150 lg/dose), the entire
suspension exposed a uniformly distributed fluorescence 23-fold higher than AH alone. Direct quantification of adsorbed antigens in vaccine formulations Standard formulations were prepared in the range of 0–150 lg/ dose and tested by the FC assay. Standard curves generated by three-parameter nonlinear regression analysis had correlation coefficients R2 > 0.99 (Fig. 5). To evaluate the reliability of the standard curves in measuring the antigen concentrations, a series of check-standard samples ranging from 3 to 100 lg/dose were assayed. The protein concentrations were determined from fluorescence readings using the y = ax3 + bx2 + cx regression equation (Table 1). The antigen concentrations calculated were in agreement with the nominal amount for both of the antigens, NadA and NHBA–GNA1030, with the overall percentage of accuracies being 85–97% for the detection range of 3.1–100 lg/dose in the monovalent vaccines and 80–97% for the detection range of 3.1–50 lg/dose in the multivalent vaccine. Interassay variation analysis showed that the coefficients of variation (CVs) of test samples for the range from 3.1 to 50 lg/dose were consistently low. The CV results obtained from triplicate samples performed in three independent assays were shown to be acceptable from 1% to 6%, indicating that the assay is highly reproducible within the described range for antigens in both the mono- and multivalent vaccines. Discussion Traditional methods for determining the protein antigen concentration of aluminum-based vaccines require the removal of the antigen from the adjuvant surface, a process that is often time-consuming. These methods expose the problems of either incomplete or partial recovery of the adsorbed proteins and modification of the protein structure during the desorption process. For these reasons, the degree of antigen adsorption to aluminumbased adjuvants is most commonly measured by centrifuging the adsorbed vaccine and assaying the supernatant for unabsorbed antigens by chromatography, colorimetric protein assays, and gel electrophoresis. A number of recent studies [17–19] have reported the direct in situ quantification of antigens; however, further exploration in this area is required before this approach can be accepted and widely used in vaccine development and manufacturing. Here we have demonstrated the usefulness of a technique that has long been used in the cell biology field as a novel application, that is, the quality control assessment of adjuvanted formulations by FC. The great advantage of the FC technology is the possibility to use the whole formulation for direct qualitative and quantitative
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Fig.4. Morphology and antigen distribution for standard formulations. (A) Representative two-dimensional dot plot indicating common morphological distribution of AH particles observed for all standard formulations (0–150 lg/dose). The gated dots appeared as a single uniform population of particles, with increased concentration of adsorbed antigens having no impact on FSC (x axis) and SSC (y axis) measurements. (B) Analysis of antigen distribution of NadA on AH aggregates using SSC and relative fluorescence intensity (RFI). The concentration of NadA in the formulation is indicated for each individual plot. The percentage of fluorescent particles increases with the concentration of NadA (0–150 lg/dose).
Fig.5. Representative standard curve for FC assay to determine antigen concentration of AH formulations. A standard curve was generated using a three-parameter nonlinear regression analysis, y = ax3 + bx2 + cx, with correlation coefficients R2 > 0.99. The y axis displays the percentage of particles with RFI > 1000. Data are the means of triplicate samples performed on three independent experiments. Error bars show standard deviations between the assays.
analysis, especially on those formulations that are based on microparticle emulsions [28] or suspensions such as the aluminumbased formulations, representing the most common platform used in licensed vaccines. The method we developed is highly accurate in determining the concentration of adsorbed proteins (85–97% in monovalent formulations, 80–97% in multivalent formulations) within a broad detection range of 3.1–100 lg/dose, with sufficient
sensitivity to detect antigen concentrations as low as 3.1 lg/dose. Furthermore, we showed that the assay is reproducible and stable, with little variations observed between formulations assayed at different times or with a time delay between the staining procedure and the FC analysis of the samples. In addition to the accuracy, sensitivity, and reproducibility, we believe that the FC assay possesses a number of other key advantages that originate from the fact of analyzing physical particles; FC detects the antigen directly on the surface of the aluminum particles. First, the degree of antigen adsorption to aluminum is measured directly without the need to rely on indirect measurements of unadsorbed antigen in the formulation of supernatants. Second, because the assay is antibody based, it is possible to use FC to monitor antigen stability while still bound to the surface of aluminumcontaining adjuvants. This could be particularly powerful for antigens where protective epitopes have been well defined and where monoclonal antibodies, specific for these regions, could be used in the FC assay to monitor the structural integrity of the epitopes over time or to monitor vaccine consistency prior to their release. Notably, a clear advantage of this assay is the ability to determine protein concentrations on each aluminum adjuvant particle, giving insight to the antigen distribution within the formulation. With guidelines in the literature [29] describing different adsorption strategies for the production of multicomponent vaccines, FCbased assays could help to monitor the uniformity of resulting vaccines and the importance of antigen distribution for efficacy. Finally, another common advantage for antibody-based assays specific for a target antigen is the potential of the assay to be
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Table 1 Results from check-standard samples for NadA (A) and NHBA-GNA1030 (B) in monovalent vaccine and for NadA (C) and NHBA-GNA1030 (D) in multivalent vaccine. Nominal concentration (lg)
Concentration detected (lg)
Accuracy (%)
(A) NadA concentrations of monovalent formulated vaccine 3.1 3.0 ± 0.1 94.8 ± 2.1 6.2 6.3 ± 0.3 96.4 ± 2.8 12.5 12.8 ± 0.2 97.6 ± 1.6 25.0 24.9 ± 0.8 97.5 ± 2.0 50.0 50.5 ± 2.8 94.8 ± 1.5 100.0 113.9 ± 12.3 84.7 ± 10.1
CV (%) 4.8 4.8 1.6 3.4 5.7 10.8
(B) NHBA-GNA1030 concentrations of monovalent formulated vaccine 3.12 2.99 ± 0.1 95.94 ± 3.92 6.25 5.69 ± 0.3 91.09 ± 5.52 12.5 11.26 ± 0.4 90.11 ± 2.86 25 23.53 ± 0.4 94.13 ± 1.66 50 46.57 ± 0.9 93.15 ± 1.87 100 107.57 ± 1.1 92.43 ± 1.11
4.08 6.07 3.17 1.76 2.00 1.03
(C) NadA concentrations of multivalent formulated vaccine 3.12 3.07 ± 0.11 97.22 ± 2.06 6.25 6.43 ± 0.13 97.17 ± 2.03 12.5 13.11 ± 0.6 95.09 ± 4.81 25 23.31 ± 0.55 93.25 ± 2.19 50 40.59 ± 1.7 81.19 ± 3.39 100 135.61 ± 24.61 64.39 ± 24.61
3.58 1.97 4.59 2.35 4.18 18.15
(D) NHBA-GNA1030 concentrations of multivalent formulated vaccine 3.12 2.99 ± 0.18 95.94 ± 5.7 6.25 6.16 ± 0.4 95.57 ± 3.76 12.5 12.16 ± 0.24 97.31 ± 1.9 25 22.65 ± 0.55 90.61 ± 2.21 50 40.16 ± 2.04 80.31 ± 4.08 100 127.61 ± 8.74 72.39 ± 8.74
[5]
[6]
[7]
[8] [9]
[10]
[11]
[12]
[13]
[14] 5.94 6.45 1.95 2.44 5.08 6.85
Note: All of the results were obtained from triplicate samples representing three independent experiments. Check-standard samples of antigens formulated with AH were freshly prepared and diluted to a final concentration with AH suspension (1500 lg/dose). Back calculations were performed by converting the percentage of fluorescent particles measured for the check-standard concentrations of antigen using three-parameter nonlinear regression analysis. % Accuracy is the percentage of similarity between the amount of antigens calculated by back calculation (detected by FC assay) and the known amount of antigens. % CV is the coefficient of variation between assays and is calculated as the standard deviation over the mean.
applied directly to complex formulations containing multiple antigens and adjuvants such as combination vaccines. To conclude, we have demonstrated that FC is a powerful tool for the characterization of AH-based vaccine formulations. Our FC-based assay is antigen specific, sensitive, and reproducible. The assay has several other potential applications, including monitoring long-term antigen stability, distribution without antigen desorption, and the possibility to extend the analysis to other adjuvants and delivery systems such as emulsions, polymeric microparticles, and liposomes.
[15]
[16]
[17]
[18]
[19]
[20]
[21] [22]
[23]
[24]
Acknowledgments The authors thank Rino Rappuoli, global head (research) of Novartis Vaccines and Diagnostics, for support in this work. The authors also acknowledge the support provided by Michele Pallaoro.
[25]
[26]
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