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Adsorption onto aluminum hydroxide adjuvant protects antigens from degradation
Vaccine xxx (xxxx) xxx
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Adsorption onto aluminum hydroxide adjuvant protects antigens from degradation Annalisa Colaprico a,⇑, Silvia Senesi a, Francesca Ferlicca a, Brunella Brunelli a, Mildred Ugozzoli b, Michele Pallaoro b, Derek T. O’Hagan a a b
GSK, Siena, Italy Novartis Vaccines and Diagnostics srl, (a GSK Company), Siena, Italy
a r t i c l e
i n f o
Article history: Received 24 September 2019 Received in revised form 31 January 2020 Accepted 2 February 2020 Available online xxxx Keywords: Aluminum hydroxide Antigen adsorption Vaccine Calorimetry capture ELISA Neisseria meningitidis type B
a b s t r a c t Aluminum based adjuvants are widely used in commercial vaccines, since they are known to be safe and effective with a variety of antigens. The effect of antigen adsorption onto Aluminum Hydroxide is a complex area, since several mechanisms are involved simultaneously, whose impact is both antigen and formulation conditions dependent. Moreover, the mode of action of Aluminum Hydroxide is itself complex, with many mechanisms operating simultaneously. Within the literature there are contrasting theories regarding the effect of adsorption on antigen integrity and stability, with reports of antigen being stabilized by adsorption onto Aluminum Hydroxide, but also with contrary reports of antigen being destabilized. With the aim to understand the impact of adsorption on three recombinant proteins which, following in vivo immunization, are able to induce functional bactericidal antibodies against Neisseria meningitidis type B, we used a range of physico-chemical tools, such as DSC and UPLC, along with in vitro binding of antibodies that recognize structural elements of the proteins, and supported the in vitro data with in vivo evaluation in mice studies. We showed that, following exposure to accelerated degradation conditions involving heat, the recombinant proteins, although robust, were stabilized by adsorption onto Aluminum Hydroxide and retain their structural integrity unlike the not adsorbed proteins. The measure of the Melting Temperature was a useful tool to compare the behavior of proteins adsorbed and not adsorbed on Aluminum Hydroxide and to predict protein stability. Ó 2020 Elsevier Ltd. All rights reserved.
1. Introduction Aluminum Hydroxide (AH) is the most widely used adjuvant in human vaccine, it is a potent enhancer of antibody production by creating a local inflammatory environment in the injection site, which activate innate immune cells. Adsorption onto the surface of AH is mostly driven by ligand exchange, electrostatic forces, hydrophobic interactions. There are no guidelines specifying if the antigen needs to be adsorbed on AH, however it is demonstrated that adsorption is necessary to exert AH effects [1]. In in vitro experiments was observed that antigen internalization by dendritic cells was enhanced when the antigen remained adsorbed on AH and that the degree of adsorption in interstitial fluid rather than the degree of adsorption in the vaccine correlates with antibody production [2]. Surely, it’s important that the adsorption rate remains constant; indeed, adsorption degree is a quality control factor for final product as an indicator for the consistency of ⇑ Corresponding author. E-mail address: [email protected] (A. Colaprico).
vaccine manufacturing processes. If the role of AH in immune response is well documented, its effect on antigen stability is still under debate. There are conflicting theories [3–12] describing to which extent AH protects or destabilizes antigens. Protein stability could be affected by structural changes, probably protein unfolding, which occur after the adsorption onto aluminum adjuvant particles [13,14]. These effects are time dependent as a result of vaccine aging and, in some cases, with significant magnitude [15], but it is not clear if they are reversible or irreversible and if there is a detrimental effect on immunogenicity. To have an accurate answer it’s necessary the integration of data from different and orthogonal techniques. A setback for assessing vaccine stability is the fact that many vaccines possess a specific biological activity that cannot be fully characterized by physico-chemical methods alone. Biological assays play an important role in the quality control of vaccine and are essential parameters of vaccine quality [16]. Ideally, a stability-indicating parameter should reflect the link between vaccine quality and efficacy, as demonstrated by immunogenicity and subsequent protective effect [17]. The aim of our study is to understand if the adsorption of antigens on AH
https://doi.org/10.1016/j.vaccine.2020.02.001 0264-410X/Ó 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: A. Colaprico, S. Senesi, F. Ferlicca et al., Adsorption onto aluminum hydroxide adjuvant protects antigens from degradation, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.001
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induces antigen protection or destabilization. We chose to focus our analysis on three antigens of the Meningococcal Serogroup B (4CMenB) vaccine [18] as a model for protein based vaccines, with the purpose to extend the method of analysis to different vaccines. We first analyzed by nano DSC antigens adsorbed (formulated antigens) and not adsorbed (unformulated antigens) on AH to compare their thermal stability. We then performed a forced degradation study on 4CMenB antigens both formulated and unformulated with AH to verify the role of the adjuvant on antigens stability and functionality. It’s difficult to characterize with antibodies the structure and stability of proteins when they are adsorbed onto the surface of aluminum salts due to steric hindrance of AH aggregates [19]. In our study we utilized a desorption method which enabled the analysis of antigens desorbed from AH in their folded form, as suggested by the SE-UPLC (SEC) analysis. This allowed the introduction of SEC as a tool to analyze desorbed antigens and of capture ELISA assay which, based on bactericidal polyclonal antibodies, could be a link between in vitro and in vivo assays. We identified important differences between the antigens thermally stressed after adsorption on AH and antigens stressed in absence of AH. We found that AH has an important protective effect on 4CMenB vaccine antigens, increases their Melting Temperature and assures their in vivo functionality also when stressed at the high temperatures tested. 2. Materials and methods 2.1. Antigens description Clinical grade material of the 4CMenB vaccine recombinant proteins was obtained from GSK Vaccines in Siena (Italy). Factor H binding protein (fHbp) fused with GNA2091, the antigens Neisseria adhesin A (NadA), the Neisseria Heparin-Binding antigen (NHBA) fused with GNA1030, were used for the study. FHbp is a surface exposed lipoprotein which induces high levels of bactericidal antibodies also protective in vivo, following passive immunization [20,21]. To enhance immunogenicity, it is fused with an accessory protein Genome derived Neisseria Antigen (GNA) 2091, another antigen identified by reverse vaccinology. NadA induces a strong bactericidal response, and is protective by passive immunization in the infant rat model [22,23]. NHBA is a lipoprotein that binds heparin, induces bactericidal antibodies in mice and is protective by passive immunization in infant rat model [24,25]. In the vaccine it is fused with an accessory protein GNA1030. The theoretical molecular weights confirmed by experimental mass spectrometry were 35 kDa for NadA, 68 kDa for NHBA-GNA1030 and 46 for fHbp-GNA2091, while isoelectric points (pI) were theoretically calculated as 4.6, 5.1, and 9.0, respectively. 2.2. Nano Differential Scanning Calorimeter (nano DSC) FHbp-GNA2091, NadA, and NHBA-GNA1030 were loaded separately in a TA instrument nano Differential Scanning Calorimeter (nano DSC) at a concentration of 0.1 mg/ml in formulation buffer (10 mM Histidine pH 6.3, 6.25 mg/ml NaCl, 2% sucrose) and when adsorbed on AH (10 mM Histidine pH 6.3, 6.25 mg/ml NaCl, 2% sucrose, 3 mg/ml AH) to monitor the differences in the amount of heat absorbed by antigens and an equal amount of matching buffer. The buffer baseline was first obtained to establish the linearity, then each antigen was scanned. An alternation of 10 cooling and heating cycles were performed from a starting temperature of 10 °C to 100 °C and 10 °C to 120 °C for NHBA-GNA1030, with a 1 °C/ s scan rate. To prevent bubble formation, cells were pressurized at a constant pressure of 3 atmospheres. The differences in heat adsorption or release between the reference cell which contains
the protein buffer and the cell of the sample, give us indication of the Melting Temperature (Tm), which is the temperature at which the 50% of protein is unfolded. 2.3. Antigen formulation AH suspension used for this study was produced at GSK Vaccines (Marburg, Germany) and also used in licensed vaccine. The formulated antigens were prepared by adsorbing the three recombinant proteins at a concentration of 0.1 mg/ml on AH (3 mg/ml) in 10 mM Histidine buffer (pH 6.3) and 2% sucrose. The final osmolality was adjusted to 300 ± 60 mOsm/kg using 2 M NaCl solution. The unformulated antigens were prepared by adding the three recombinant proteins at a concentration of 0.1 mg/ml in 10 mM Histidine buffer (pH 6.3) and 2% sucrose. The final osmolality was adjusted to 300 ± 60 mOsm/kg using 2 M NaCl solution. For the nano DSC analysis and for the in vivo study, the antigens were formulated individually. The formulations were stirred for 15 min at room temperature (RT), stored overnight (17 h) at 4 °C and analyzed the day after. The entire panel of analysis was repeated three times. 2.4. Thermal stress The formulated antigens and the unformulated antigens were split into 5 doses: one dose was not stressed (NS), the other ones were stored 17 h at 37 °C or incubated for 3 h at 50 °C, 65 °C and 80 °C. After the stress the samples were cooled to RT and immediately processed for the analysis. 2.5. Antigens recovery from Aluminum Hydroxide The antigens recovery consisted on the desorption of antigens from AH upon exposure to high concentration of phosphate buffer. Formulated antigens were mixed to a double volume of desorbing buffer (450 mM KH2PO4 pH 6.5, 15% Ethylene glycol, 0.02% Tween 80) in a 2 ml Eppendorf Protein LoBind Tube and incubated with gentle rocking agitation at 25 °C for 17 h. After incubation, the tubes were centrifuged at 2100g for 20 min. The supernatants were then removed and analyzed by Capture ELISA, RP-UPLC, SE-UPLC and SDS-PAGE. The unformulated antigens were treated with the same desorption procedure. 2.6. Reverse-Phase Ultra Performance Liquid Chromatography (RPUPLC) After the stress and the desorption procedure, formulated antigens and unformulated antigens were analyzed for protein content using an Acquity UPLC Waters chromatographic system equipped with a photodiode array detector (PDA, Waters). The samples obtained after the desorption procedure (10 ll), with a theoretical concentration of 33 mg/ml, were loaded on a C4 BEH300 column 1.7 mm (2.1 mm ID 150 mm, Waters) and antigens were eluted with a conventional RP-UPLC system utilizing a mobile phase of water/0.1% TFA and acetonitrile/0.1% TFA at a flow rate of 0.6 ml/ min. A standard curve (200–6.25 mg/ml) was prepared by twofold serial dilution of a stock of 200 mg/ml of three 4CMenB antigens in the formulation buffer. To monitor potential effects of the elution method on the antigens, a sample with the same formulation composition was treated with desorbing buffer in the same manner of formulated and unformulated antigens and used as reference control. The samples were loaded onto column and eluted with a gradient of acetonitrile (20–80%) and quantified by peak area calculation at 214 nm wavelength. Standards were analyzed both at the beginning and at the end of each analytical session.
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2.7. Size Exclusion Ultra Pressure Liquid Chromatography (SE-UPLC)
2.11. Antigen-Specific Capture ELISA
SE-UPLC is of extreme value for the analysis of multimeric antigen to monitor their quaternary structure and to probe the presence of aggregated protein in general. An Acquity UPLC H-class chromatographic system (Waters) equipped with a PDA detector was used for SEC separation with UV detection and spectral analysis. A BEH 200 Å column (4.6 mm ID 300 mm, 1.7 mm, Waters), at 30 °C was used, with the corresponding pre-column. The samples obtained after the desorption procedure (10 ll) were loaded onto the gel filtration column and eluted with an isocratic mobile phase: 0.5 M KH2PO4 pH 7.2 with 20% ACN, flow rate of 0.3 ml/min. To monitor the quality of the antigen, the UV absorbance at 214 nm were recorded and the chromatograms peaks were compared with each relative reference control and standard.
The reagents of the Meningococcal Antigen Typing System (MATS) [26] were used to assess the integrity of the antigens after the thermal stress. Briefly, after the desorption procedure, formulated and unformulated antigens were diluted in dilution buffer (0.1% BSA in PBS1X) to reach a final concentration of 10 ng/ml. Two-fold serial dilutions were plated in duplicate in ELISA plates, which were pre-coated with rabbit polyclonal antibodies against fHbp, NHBA, or NadA to capture the antigens. Plates were sealed and incubated for 1 h at 37 °C and then washed with PBS/0.05% Tween. After adding 100 ll of biotinylated rabbit polyclonal antibody, the plates were incubated for 1 h at 37 °C. Following the removal and wash of the well, 100 ll of streptavidin-HRP was added and incubated for 30 min at 37 °C. The wells were developed with o-Phenylenediamine dihydrochloride (OPD, Sigma) for 20 min at RT, the reaction was stopped by addition of 50 ll of 4 N H2SO4 and read immediately at 492 nm with an ELX 808 Biotek plate reader. A standard curve created by 2-fold dilution of a 10 ng/ml antigen stock was included on each plate. As internal quality control and to monitor the inter-assay variability, antigen at a fixed concentration (2.5 ng/ml) was plated.
2.8. SDS-PAGE Reducing SDS-PAGE was performed using NuPAGE pre-cast 4– 12% Bis-Tris Gels (Invitrogen). Aliquots of desorbed antigens (20 ll) were mixed with 4X Loading Sample Buffer (LSB) (SDS 8%, Glycerol 25%, Bromophenol blue 0.25%, DTT 6%, Tris-HCl 0.25 M pH 6.8), heated at 90 °C for 10 min and loaded into the gel. To evaluate the amount of antigens not desorbed from AH, the pellet obtained after the desorption procedure was resuspended with 0.6 M KH2PO4, 5% SDS and treated 10 min at 100 °C. The samples were centrifuged 20 min at 2100g, 20 ll of the obtained supernatant were loaded in the gel. To evaluate the amount of antigens not adsorbed on AH at the different stress temperatures, 200 ll of stressed formulations were centrifuged at 2100g for 20 min and the supernatants obtained were treated with DOC 0.05% and TCA 4.8%. After 10 min were centrifuged at 2100g for 20 min and the pellets were resuspended with 10 ll of TrisHCl pH 8 and 10 ll of LSB, heated at 90 °C for 10 min and loaded into the gel. The molecular weight standard (Seeblue plusInvitrogen) was loaded on each gel for reference. The proteins were electrophoretically separated using the Invitrogen Xcell SureLockTm Mini-Cell system (Invitrogen), stained with Easyblue Invitrogen and de-stained with milliQ Water. Gel images were then digitalized on GelDoc Biorad. 2.9. Ethical statement All animal studies were carried out in compliance with Italian legislation in force at that time, on the care and use of animals in experimentation (Legislative Decree 64/2006-A) and with Novartis Animal Welfare Policy and Standards. Protocols were approved by the Italian Ministry of Health (authorization AEC/2011–01) and by local Novartis Animal Welfare Body 2.10. Mice immunization Groups of 8 CD1 six-weeks old female mice were immunized by the intraperitoneal route using different doses (20 and 1 lg in 200 ll) of not stressed and stressed formulated antigens. Following the 3Rs recommendations, the unformulated antigens were not analyzed in vivo because we know from undisclosed data that, in absence of AH the SBA titers are lower and not comparable with the ones obtained in presence of AH, moreover, adsorbing on AH the unformulated antigens after the thermal stress could change their structure leading to misinterpret the results. For the same reason NHBA-GNA1030 and fHbp-GNA2091 stressed at 65 °C were not analyzed in vivo because, based on nano DSC data, we didn’t expect variations in SBA titers in the samples stressed at this temperature. Animals were immunized on days 1, 21 and 35 and the sera were collected at day 49.
2.12. Serum bactericidal antibody assay Serum bactericidal activity against N. meningitidis strains was evaluated as previously described [27] with pooled baby rabbit serum (Cederlane Laboratories). Briefly, bacteria were grown in MH (Muller Hinton) broth plus 0.25% glucose for approximately 1.5 h at 37 °C with shaking until early log phase (OD600) and then diluted in Dulbecco’s buffer (SIGMA) plus 1% BSA and 0.1% Glucose (DPBS) to approximately 104–105 CFU/ml. Serum bactericidal titers were defined as the serum dilution resulting in a 50% decrease in the CFU/ml after 60 min of incubation of bacteria with the reaction mixture, compared to the control. 3. Results 3.1. Nano DSC analysis of 4CMenB antigens Melting Temperatures Calorimetry is an unsurpassed tool for measuring the thermal properties of materials to establish a connection between temperature and specific physical properties of substances [28]. It is the ideal technique to study protein stability and the reversibility of thermal processes. It was also used to determine thermogram profile before and after desorption of antigen from AH, to asses impact of desorption process on antigen stability [29]. Parameters like the shape of the thermogram and the Tm provide the most immediate information on increased or decreased stability of the protein and can be used in pre-formulation work to scout the best formulation conditions to improve protein stability. The three 4CMenB vaccine antigens were analyzed in nano DSC in order to identify their Tm when formulated or unformulated with AH. The analysis of antigens formulated with AH showed a trend of increased Tm respect to unformulated antigens, as shown in Table 1, with the notable exception of the accessory protein GNA2091 fused with fHbp. The DSC thermogram of NadA changes dramatically upon adsorption onto AH, we in fact measured a Tm that was found to be 10 °C higher with respect to the Tm that was measured in absence of AH. Also fHbp and NHBA-GNA1030 were affected by adsorption onto the AH matrix which caused an increase of about 2 °C and 5 °C respectively, thus confirming that AH adsorption appears to stabilize these antigens. With the aim to observe antigen modifications and investigate if they correlate with alteration in bactericidal titers, we chose to stress the three proteins at the temperatures
Please cite this article as: A. Colaprico, S. Senesi, F. Ferlicca et al., Adsorption onto aluminum hydroxide adjuvant protects antigens from degradation, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.001
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Table 1 Melting temperature comparison of formulated and unformulated 4CMenB antigens. The fHbp-GNA2091 presented three peaks each belonging to a component of the fusion protein. Antigens
Portion or Domain providing signal
Tm of Unformulated Antigens
NHBA-GNA1030 fHbp-GNA2091
NHBA GNA2091 fHbp N-Term fHbp C-Term Full lenght
98 59 67 82 50
NadA
°C °C °C °C °C
Tm of Formulated Antigens 103 °C 50 °C 69 °C 84 °C 60 °C
they unfold. Based on nano DSC data we therefore decided to stress the 4CMenB antigens (i.e. NadA, NHBA-GNA1030, fHbp-GNA2091), formulated and unformulated with AH at three different stress conditions, namely 17 h at 37 °C, 3 h at 50 °C which corresponds to the Tm of unformulated NadA, 3 h at 65 °C, temperature at which, according to the Tm, formulated NadA should be denatured, and 3 h at 80 °C, which is a temperature at which we expect to find NadA totally degraded, fHbp-GNA2091 partial functional and NHBA-GNA1030 still totally functional. 3.2. RP-UPLC: Quantification of stressed unformulated and formulated antigens RP-UPLC resolves proteins with different hydrophobic properties that may have undergone significant primary structure changes. When antigens were stressed in absence of AH we observed a lower recovery after 3 h at 80 °C, probably due to precipitation (Fig. 1), while for NadA the recovery start to decrease after 3 h at 50 °C, in correspondence to its Tm. We observed that antigens recovery from AH was stress-temperature dependent, in fact, while the not stressed antigens were totally recovered from AH (Fig. 1), the desorption rate slowly decreased with increasing the stress temperature. This effect could be due to a rearrangement of antigens onto AH surface which interfere with desorption [4,30]. This hypothesis was confirmed when we analyzed the same desorbed samples by SDS-PAGE which showed that, the higher the stress temperature is, the more difficult to desorb antigens from AH (Fig. 2a). We also analyzed the residual formulation pellet obtained after 17 h of desorption, treated with harsher condition and we confirmed that, increasing the temperature, an increasing quantity of antigens remain adsorbed on AH (Fig. 2b). When analyzed 24 h after the preparation, in the formulation supernatants is usually present the fHbp-GNA2091 in a quantity <10% of the total amount and it adsorbs more strongly onto AH after storage at 4 °C. In the analysis of stressed formulations we found that increasing the temperature, to a lower recovery of antigen corresponds also a lower amount of antigen in the formulation supernatant (Fig. 2c), suggesting that it adsorbs more strongly onto AH surface, as hypothesized with RP-UPLC results. 3.3. Capture ELISA analysis of stressed unformulated and formulated antigens The ideal condition to analyze antigens alteration in a vaccine would be when still adsorbed on adjuvant. AH characteristics, such as its greater dimension respect to antigens [19] which create a steric interference to antibody accessibility, the fact that it could mask important epitopes for antibodies binding, and a possible antibodies adsorption on its surface if not well saturated, induced us to abandon the idea of a direct detection of antigens on AH, we in fact obtained variable results and we directed our investigation on antigens desorbed from AH after stress. For this reason, we
introduced a desorption procedure which didn’t alter aggregation status of proteins (see Fig. 5, not stressed samples). After being stressed on AH, desorbed antigens were analyzed also in capture ELISA to measure their capacity to be captured by specific, reproducible and standardized polyclonal antibodies coated on plates. The analysis with a biological assay based on the use of bactericidal antibodies, helped us to understand if the qualitative changes observed in SE-UPLC (Fig. 5) were so significative to alter antigen functionality. We obtained a good recovery of not stressed antigens (Fig. 3) which drastically decreased after 3 h of storage at 80 °C, when the three antigens were less recognized by antibodies (NHBA-GNA1030: 20%, fHbp-GNA2091: 3%, NadA: 4%). Differently, antibody recognition of formulated NadA starts to decrease already after 3 h of storage at 50 °C (recovery 45%) and 65 °C (recovery 10%) (Fig. 3). The analysis of unformulated antigens had the same trend of the formulated ones, except for NadA, for which we observed a complete loss of antibody recognition after 3 h at 50 °C in absence of AH, as expected based on nano DSC analysis, which indicated that the Tm of unformulated NadA was 50 °C (Table 1). This support the thesis that AH protects antigen from thermal degradation.
3.4. Ratio capture ELISA/RP-UPLC Because of we found that when the formulations were stressed at high temperatures we couldn’t totally desorb antigens from AH (Figs. 1–2), and since we analyzed in capture ELISA desorbed antigens, to avoid an underestimation of antigen functionality when stressed at higher temperatures, we considered the percentage of recovery obtained in RP-UPLC as the maximum amount of antigen we can desorb, and impose it as the 100% with which compare the recovery obtained in capture ELISA (Fig. 4). Interestingly we found that, even if NHBA-GNA1030 at 80 °C was less desorbed from AH, due probably to a rearrangement of antigen on adjuvant, it was still totally recognized by bactericidal antibody, which induced us to speculate that also in vivo the stressed formulation could still induce protection. When formulated fHbp-GNA2091 was stressed for 3 h at 65 °C, almost the totality of the desorbed antigen was still recognized by antibody. When stressed 3 h at 80 °C, only the 13% of the desorbed antigen was still captured by antibody, suggesting a decrease of functionality which we hypothesized could be found also in SBA analysis. As expected from nano DSC analysis, we observed a significative decrease in the percentage of formulated NadA still recognized by antibody after a thermal stress of 3 h at 65 °C (28%), and after being stressed 3 h at 80 °C the antigen is no more recognized by antibodies. Antibodies coated on plate are bactericidal, therefore, capture ELISA could be considered as ‘‘functional assay” for the part endowed of SBA activity and these results could predict the functionality of antigens in vivo, when stressed at these high temperatures. Unformulated antigens showed a similar trend, they differentiate from formulated antigens after 3 h at 65 °C when the protective effect of AH became more evident. For NadA we observed an important differentiation between formulated and unformulated antigen already after 3 h at 50 °C, when the unformulated antigen was no more recognized by antibody, confirming our hypothesis based on nano DSC analysis, according with which AH adsorption increases antigen Tm, in the case of NadA it increases from 50 °C in absence of AH to 60 °C when formulated with AH. These results showed that the adsorption on AH protect antigens from thermal degradation and indicated that the three vaccine antigens are very robust and resistant to high temperatures, in fact, NHBA-GNA1030 is still recognized by bactericidal antibody at 80 °C, fHbp-GNA2091 recognition decrease only at 80 °C and NadA is still intact at 50 °C. The ratio between capture ELISA and RP-UPLC provides a very useful way to understand
Please cite this article as: A. Colaprico, S. Senesi, F. Ferlicca et al., Adsorption onto aluminum hydroxide adjuvant protects antigens from degradation, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.001
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NHBA-GNA1030
% of antigen recovery
120
Unformulated Antigens Formulated Antigens
90 60 30 0 Not stressed 37°C
50°C
65°C
80°C
fHbp-GNA2091
% of antigen recovery
120
Unformulated Antigens Formulated Antigens
90 60 30 0 Not stressed 37°C
50°C
65°C
80°C
NadA % of antigen recovery
120
Unformulated Antigens Formulated Antigens
90 60 30 0 Not stressed 37°C
50°C
65°C
80°C
Fig. 1. Percentage of antigen recovery in RP-UPLC at different temperatures. The recovery of unformulated antigens decreased in correspondance of antigens Tm. The desorption rate of formulated antigens decreased with increasing the stress temperature (dotted bars), probably due to a rearrangement of antigens on Alum. Average of three experiments.
how much antigen is still somehow ‘functional’ in the recovered amount. 3.5. SE-UPLC SE-UPLC separates analytes on the basis of a combination of their hydrodynamic size, diffusion coefficient, and surface properties. It can show protein polymerization, oligomerization or degradation. It was particularly useful to understand whether changes occurred on the stressed unformulated and formulated antigens in comparison with the original untreated sample. Our desorption procedure allowed us to analyze antigens desorbed from AH in their folded form. Thermally stressed unformulated and formulated antigens desorbed from AH were injected in SE-UPLC for a
qualitative analysis. We verified that the desorption treatment didn’t cause protein unfolding and didn’t impact antigen profile (the Not stressed formulated antigen has the same profile of Standard), moreover the unformulated antigens received the same desorption treatment of the formulated antigens so that all the changes we observed in SE-UPLC were assignable to thermal effect. We observed only a decrease in peaks intensity with the increasing temperature of formulated NHBA-GNA1030 and fHbp-GNA2091 (Fig. 5), which were due to the lower recovery of antigens we have previously described (Fig. 1). We could also observe an increase of an aggregate belonging to unformulated NHBA-GNA1030 (confirmed by SE-UPLC on single antigen), which became more intense with increasing temperature, while remained constant when the antigen was adsorbed on AH. There is a conformational change of
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(a)
NS
37 °C 50 °C 65 °C 80 °C
(b)
NS 37 °C 50 °C 65 °C 80 °C
(c)
NS
37 °C
50 °C 65 °C 80°C
NHBA-GNA1030
fHbp-GNA2091 NadA
Ags desorbed ON
Ags residues on AH
Ags in the Supernatant
Fig. 2. SDS-PAGE of desorbed antigens (a), antigens residues still adsorbed on AH after desorption (b) and antigens in formulation supernatant (c). SDS-PAGE confirms RPUPLC results: the higher the temperature is, the less we recover antigens from AH; they remain adsorbed on it, probably due to rearrangement phenomena.
unformulated NadA in correspondence of Tm (50 °C) in fact, increasing the temperature, there is a progressive prevalence of the monomeric form (confirmed by SE-UPLC on single antigen) respect to the trimeric form. The two forms are well discriminated in capture ELISA (Figs. 3–4), in fact the monomeric form is no more recognized by antibody. On the other side, when formulated with AH, NadA remains in its trimeric form at 50 °C and become monomer only at 65 °C. Results clearly indicated that the three antigens are stabilized by the adsorption on AH. 3.6. Serum bactericidal antibody levels (SBA) of stressed formulations SBA is the most important method for measuring functional activity of serum antibodies against meningococcus [31–35]. To evaluate immunogenicity and functionality in vivo, CD1 mice were immunized with monovalent stressed and not stressed formulations at different antigens doses. Complement-mediated serum bactericidal antibody titers was measured 14 days after the third immunization. The bactericidal activity of sera resulting from mice immunized with stressed and not stressed formulated NHBAGNA1030 was tested against two strains, M4407 and M3279, respectively highly and lowly expressing the serobase protein, as estimated by Meningococcal Antigen Typing System (MATS) [26] (Table 2A). Thermally stressed formulated NHBA-GNA1030 had the same serum bactericidal titers of not stressed formulation, in agreement with what we observed in capture ELISA/RP-UPLC ratio. Sera of mice immunized with stressed formulated fHbp-GNA2091 were tested against the strains MC58 and M01-0240660 (UK660), respectively highly expressing and lowly expressing the protein by MATS. In UK660, 1 mg of antigen induced similar SBA titers even after a stress of 3 h at 50 °C. When the stress was for 3 h at 80 °C SBA titers decreased by 3 folds, confirming what we had predicted analyzing capture ELISA/UPLC ratio (Table 2B). With 20 mg of fHbpGNA2091 we reached the plateau, so it was not possible to appreciate differences in SBA titers when the formulation was stressed at 80 °C. The serum bactericidal activity of mice immunized with formulated NadA was tested against the strains 5/99 and 2996,
respectively highly expressing and lowly expressing the protein by MATS. As expected, based on capture ELISA data, bactericidal activity decreased after a stress of 3 h at 65 °C (Table 2C), which is a temperature slightly higher than NadA Tm (60 °C) when adsorbed on AH (Tab. 1). It is very evident how the presence of AH confers stability to NadA which, when adsorbed on AH, is still functional after a stress of 3 h at 50 °C while, in absence of adjuvant, at the same temperature is unfolded (Fig. 5) and no more recognized by bactericidal antibody (Fig. 3). These results are in complete agreement with what we predicted basing on the ratio capture ELISA/RP-UPLC (Fig. 4). 4. Discussion While the role of AH as immune potentiator is well documented, its role on antigen stability is still under discussion. For this reason we analyzed three antigens of the 4CMenB vaccine both formulated and unformulated with AH to verify if the adsorption on adjuvant stabilizes or destabilizes antigens. By nano DSC analysis we found that, when adsorbed onto AH surface, the Melting Temperature increased from 2 to 10 °C, depending on antigen. We performed a forced degradation study to deliberately alter either formulated or unformulated antigens till their end-point identified by nano DSC and cause alteration in immune response measured by Serum Bactericidal Assay. We observed that, when antigens were thermally stressed adsorbed on AH, they rearranged or precipitate on its surface and at high temperature the adsorption became stronger, so that was difficult to desorb them from AH. In RP-UPLC we had a lower recovery of antigens in correspondence of higher stress temperature, and in SE-UPLC we observed a peak intensity decrease. The analysis of desorbed antigens in capture ELISA indicated that the three 4CMenB vaccine antigens were very robust and stable, in fact, only elevated temperature of stress induced decrease of antibodies recognition. Because of antibodies coated on capture ELISA plates were bactericidal, we hypothesized that we could obtain the same results in vivo, in fact, the analysis of sera bactericidal activity measured by SBA, totally confirmed the results of capture ELISA. On the other side, when antigens were
Please cite this article as: A. Colaprico, S. Senesi, F. Ferlicca et al., Adsorption onto aluminum hydroxide adjuvant protects antigens from degradation, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.001
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NHBA-GNA1030
% of antigen recovery
120
Unformulated Antigens Formulated Antigens
90 60 30 0 Not stressed
37 °C
50 °C
65 °C
80 °C
fHbp-GNA2091
% of antigen recovery
120
Unformulated Antigens Formulated Antigens
90 60 30 0 Not stressed 37 °C
50 °C
65 °C
80 °C
NadA
% of antigen recovery
120
Unformulated Antigens Formulated Antigens
90 60 30 0 Not stressed
37 °C
50 °C
65 °C
80 °C
Fig. 3. Percentage of antigens recovery in capture ELISA plates. Comparison between stressed formulated antigens (dotted bars) and stressed unformulated antigens (black bar). There is a great impact of AH on NadA stability: after 3 h at 50 °C the unformulated NadA is no more recognized by antibodies, while when formulated with AH loses its recognition only after 3 h at 65 °C, as predicted by nano DSC analysis. Average of three experiments.
stressed in absence of AH, were more sensible to thermal stress, in SE-UPLC we observed an increase of an aggregate belonging to NHBA-GNA1030 which became more intense when temperature increased. In absence of AH and in correspondence of its Tm NadA became a monomer, while when stressed at the same temperature when adsorbed on AH, remained in its trimeric form which, unlike the monomer, is still recognized by bactericidal antibody. Results demonstrated that AH has an important effect of protection on the three investigated antigens, limiting the thermal degradation in correspondence of their respective Tm. The mechanisms of protection are complex and do not follow a general rule. Adsorption on a highly charged surface like that of AH has the potential to change the stability, the flexibility and the hydration shell of the
antigen. Antigen will react to adsorption onto AH by finding a new folding state dictated by the equilibrium between the external adsorption forces competing with the internal stabilizing interactions and this new equilibrium could be one key possible mechanism leading to antigen stabilization/destabilization upon AH adsorption. If the adsorption derived forces will not be able to impact the quaternary/tertiary organization of the antigen, this will likely be trapped in a state where it still retains important folding features. At the same time, the interaction with the adjuvant will also reduce the antigen accessibility to denaturing agents/stress resulting in an increased stabilization and functionality, as seen in our data. The different proteins adsorption and stability profiles explain the variable results reported in literature,
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Ratio Capture ELISA/RP
NHBA-GNA1030
Formulated Antigens
100
Unformulated antigens
80 60 40 20 0 Not stressed 37 °C
50 °C
65 °C
80 °C
Ratio Capture ELISA/RP
fHbp-GNA2091
100
Formulated Antigens Unformulated antigens
80 60 40 20 0 Not stressed 37 °C
50 °C
65 °C
80 °C
Ratio Capture ELISA/RP
NadA Formulated Antigens
100
Unformulated antigens 80 60 40 20 0 Not stressed 37 °C
50 °C
65 °C
80 °C
Fig. 4. Representation of the ratio capture ELISA/RP-UPLC. In the figure is reported the percentage of desorbed formulated antigens (solid line) and unformulated antigens (dotted line) still recognized by antibodies after thermal stress. AH has a protective effect on antigens, above all on NadA which at 50 °C is still functional when adsorbed on AH Average of three experiments.
highlighting the need to study the matter case by case. We can conclude that AH has a role not only as enhancer of antibody production or as antigen depot, it also has the potential to preserves antigens integrity, increasing their stability and protecting them from thermal degradation. Contributions All authors reviewed and approved the final version of the manuscript. AC, MU, MP: conceiving and designing the experiments. AC: performing experiments and writing the manuscript. AC, MU, MP, DOH: analyzing data and interpreting experiments results. SS: setting up the Desorption method and the SE-UPLC method.
BB and FF: setting up, performing and interpreting SBA analysis. AC, SS, FF, BB, MU, MP, DOH: critically reviewing the manuscript. Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: This work was sponsored by Novartis Vaccines and Diagnostics Srl (now part of the GSK group of companies) which was involved in all stages of the study conduct and analysis. AC, SS, FF, BB, MU, MP and DOH were employees of Novartis Vaccines and Diagnostics Srl at the time of the study (now part of the GSK group of companies). AC, SS, FF, BB and DOH are now employees of the GSK group of companies.
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Unformulated Antigens
Formulated Antigens
0.040
0.040
NHBAGNA1030
0.030
NHBAGNA1030
0.030
NadA 0.020
0.010
0.020
0.010
0.000
0.000
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
10.50
11.00
11.50
12.00
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
Minutes
fHbpGNA2091
10.50
11.00
11.50
12.00
11.00
11.50
12.00
11.00
11.50
12.00
fHbpGNA2091
NadA
0.020
AU
AU
10.00
NHBAGNA1030
0.030
NadA
0.010
0.020
0.010
0.000
0.000
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
10.50
11.00
11.50
12.00
5.00
Minutes
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
10.50
Minutes
0.040
0.040
0.030
NadA
NHBAGNA1030
fHbpGNA2091
fHbpGNA2091
NadA
NadA monomer
0.020
NHBAGNA1030
0.030
AU
AU
9.50
0.040
NHBAGNA1030
0.030
3h 50 °C
9.00
Minutes
0.040
ON 37 °C
fHbpGNA2091
NadA AU
AU
Not stressed
fHbpGNA2091
0.010
0.020
0.010
0.000
0.000
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
10.50
11.00
11.50
12.00
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
Minutes
9.00
9.50
10.00
10.50
Minutes
0.040 0.040
NHBAGNA1030
0.030
fHbpGNA2091
NHBAGNA1030
0.030
0.020
0.020
NadA monomer
0.010
0.010
0.000
0.000
5.00
5.00
fHbpGNA2091
NadA AU
3h 65 °C
AU
NadA
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
10.50
11.00
11.50
5.50
6.00
6.50
7.00
7.50
8.00
12.00
8.50
9.00
9.50
10.00
10.50
11.00
11.50
12.00
11.00
11.50
12.00
Minutes
Minutes 0.040
0.040
fHbpGNA2091
NHBAGNA1030
0.030
0.030
NadA
0.020 AU
AU
NadA
3h 80 °C
0.010
fHbpGNA2091
NHBAGNA1030
0.020
0.010
0.000 0.000
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
10.50
11.00
11.50
12.00
5.00
5.50
6.00
6.50
7.00
7.50
8.00
Minutes
8.50
9.00
9.50
10.00
10.50
Minutes
Fig. 5. Formulated antigens (in green) and unformulated antigens (in orange) profile in SE-UPLC after thermal stress, compared to the antigens Standard (in black). The thermal degradation of formulated antigens is slower respect to the degradation of unformulated antigens. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 2 Bactericidal titers elicited in mice by stressed and not stressed (N.S.) formulated antigens against antigen-specific strains. In (A) is shown SBA titers against NHBA-GNA1030. In both strains and for both doses tested there were no differences between stressed and not stressed vaccine. In (B) we observed a decrease of SBA titers against fHbp-GNA2091 in UK660 strain, when the formulation with 1 mg of antigen was stressed 3 h at 80 °C. In (C) is visible an important decrease of SBA titers against NadA after 3 h at 65–80 °C. All these results are comparable to the ratio capture ELISA/RP-UPLC (Fig. 4). (A) NHBA-GNA1030 Tested strain
M4407
Tested strain
M3279
Stress conditions
N.S.
37 °C
50 °C
80 °C
Stress conditions
N.S.
37 °C
50 °C
80 °C
SBA titers with 20 mg Ag SBA titers with 1 mg Ag
16384 16384
16384 2048
16384 16384
16384 16384
SBA titers with 20 mg Ag SBA titers with 1 mg Ag
1024 2048
4096 512
2048 4096
1024 2048
(B) fHbp-GNA2091 Tested strain
MC58
Tested strain
M01 0240660/UK660
Stress conditions
N.S.
37 °C
50 °C
80 °C
Stress conditions
N.S.
37 °C
50 °C
80 °C
SBA titers with 20 mg Ag SBA titers with 1 mg Ag (C) NadA
65536 65536
65536 65536
65536 65536
16384 16384
SBA titers with 20 mg Ag SBA titers with 1 mg Ag
16384 16384
8192 16384
16384 8192
8192 1024
Tested strain
5/99
Tested strain
2996
Stress conditions
N.S.
37 °C
50 °C
65 °C
80 °C
Stress conditions
N.S.
37 °C
50 °C
65 °C
80 °C
SBA titers with 20 mg Ag SBA titers with 1 mg Ag
16384 16384
16384 16384
16384 32768
256 512
256 256
SBA titers with 20 mg Ag SBA titers with 1 mg Ag
1024 256
2048 512
2048 256
128 256
256 128
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Acknowledgements The authors thanks Marzia M. Giuliani for allowing the use of MATS kit for the execution of the capture ELISA assay, applied to assess the integrity of antigens after the thermal stress. References [1] Mannhalter JW et al. Modulation of the human immune response by the nontoxic and non-pyrogenic adjuvant aluminium hydroxide: effect on antigen uptake and antigen presentation. Clin Exp Immunol 1985;61(1):143–51. [2] Morefield GL et al. Role of aluminum-containing adjuvants in antigen internalization by dendritic cells in vitro. Vaccine 2005;23(13):1588–95. [3] Zheng Y et al. The structural stability of protein antigens adsorbed by aluminium hydroxide in comparison to the antigens in solutions. Spectroscopy 2007;21(5–6):257–326. [4] Jones LS et al. Effects of adsorption to aluminum salt adjuvants on the structure and stability of model protein antigens. J Biol Chem 2005;280(14):13406–14. [5] Braun LJ et al. The role of adjuvant in mediating antigen structure and stability. J Pharm Sci 2012;101(4):1391–9. [6] Dong A et al. Secondary structures of proteins adsorbed onto aluminum hydroxide: Infrared spectroscopic analysis of proteins from low solution concentrations. Anal Biochem 2006;351(2):282–9. [7] Agopian A et al. Secondary structure analysis of HIV-1-gp41 in solution and adsorbed to aluminum hydroxide by Fourier transform infrared spectroscopy. Biochim Biophys Acta 2007;1774(3):351–8. [8] Iyer V et al. Preformulation characterization of an aluminum salt-adjuvanted trivalent recombinant protein-based vaccine candidate against Streptococcus pneumoniae. J Pharm Sci 2012;101(9):3078–90. [9] Peek L et al. Effects of stabilizers on the destabilization of proteins upon adsorption to aluminum salt adjuvants. J Pharm Sci 2007;96(3):547–57. [10] Ausar SF. Application of extrinsic fluorescence spectroscopy for the high throughput formulation screening of aluminum-adjuvanted vaccines. J Pharm Sci 2011;100(2):431–40. [11] Ljutic B et al. Formulation, stability and immunogenicity of a trivalent pneumococcal protein vaccine formulated with aluminum salt adjuvants. Vaccine 2012;30(19):2981–8. [12] Regnier M et al. Structural perturbation of diphtheria toxoid upon adsorption to aluminium hydroxide adjuvant. Vaccine 2012;30(48):6783–8. [13] Norde W. Adsorption of proteins from solution at the solid-liquid interface. Adv Colloid Interface Sci 1986;25:267–340. [14] Rabe M et al. Understanding protein adsorption phenomena at solid surfaces. Adv Colloid Interface Sci 2011;162(1–2):87–106. [15] Hem SL et al. Preformulation studies–The next advance in aluminum adjuvant-containing vaccines. Vaccine 2010;28(31):4868–70. [16] Xing D et al. Laboratory testing of whole cell pertussis vaccine: a WHO proficiency study using the Kendrick test. Vaccine 2001;20(3–4):342–51. [17] Dobbelaer R et al. Guidelines on stability evaluation of vaccines. Biologicals 2009;37(6):424–34.
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Vaccine journal homepage: www.elsevier.com/locate/vaccine
Adsorption onto aluminum hydroxide adjuvant protects antigens from degradation Annalisa Colaprico a,⇑, Silvia Senesi a, Francesca Ferlicca a, Brunella Brunelli a, Mildred Ugozzoli b, Michele Pallaoro b, Derek T. O’Hagan a a b
GSK, Siena, Italy Novartis Vaccines and Diagnostics srl, (a GSK Company), Siena, Italy
a r t i c l e
i n f o
Article history: Received 24 September 2019 Received in revised form 31 January 2020 Accepted 2 February 2020 Available online xxxx Keywords: Aluminum hydroxide Antigen adsorption Vaccine Calorimetry capture ELISA Neisseria meningitidis type B
a b s t r a c t Aluminum based adjuvants are widely used in commercial vaccines, since they are known to be safe and effective with a variety of antigens. The effect of antigen adsorption onto Aluminum Hydroxide is a complex area, since several mechanisms are involved simultaneously, whose impact is both antigen and formulation conditions dependent. Moreover, the mode of action of Aluminum Hydroxide is itself complex, with many mechanisms operating simultaneously. Within the literature there are contrasting theories regarding the effect of adsorption on antigen integrity and stability, with reports of antigen being stabilized by adsorption onto Aluminum Hydroxide, but also with contrary reports of antigen being destabilized. With the aim to understand the impact of adsorption on three recombinant proteins which, following in vivo immunization, are able to induce functional bactericidal antibodies against Neisseria meningitidis type B, we used a range of physico-chemical tools, such as DSC and UPLC, along with in vitro binding of antibodies that recognize structural elements of the proteins, and supported the in vitro data with in vivo evaluation in mice studies. We showed that, following exposure to accelerated degradation conditions involving heat, the recombinant proteins, although robust, were stabilized by adsorption onto Aluminum Hydroxide and retain their structural integrity unlike the not adsorbed proteins. The measure of the Melting Temperature was a useful tool to compare the behavior of proteins adsorbed and not adsorbed on Aluminum Hydroxide and to predict protein stability. Ó 2020 Elsevier Ltd. All rights reserved.
1. Introduction Aluminum Hydroxide (AH) is the most widely used adjuvant in human vaccine, it is a potent enhancer of antibody production by creating a local inflammatory environment in the injection site, which activate innate immune cells. Adsorption onto the surface of AH is mostly driven by ligand exchange, electrostatic forces, hydrophobic interactions. There are no guidelines specifying if the antigen needs to be adsorbed on AH, however it is demonstrated that adsorption is necessary to exert AH effects [1]. In in vitro experiments was observed that antigen internalization by dendritic cells was enhanced when the antigen remained adsorbed on AH and that the degree of adsorption in interstitial fluid rather than the degree of adsorption in the vaccine correlates with antibody production [2]. Surely, it’s important that the adsorption rate remains constant; indeed, adsorption degree is a quality control factor for final product as an indicator for the consistency of ⇑ Corresponding author. E-mail address: [email protected] (A. Colaprico).
vaccine manufacturing processes. If the role of AH in immune response is well documented, its effect on antigen stability is still under debate. There are conflicting theories [3–12] describing to which extent AH protects or destabilizes antigens. Protein stability could be affected by structural changes, probably protein unfolding, which occur after the adsorption onto aluminum adjuvant particles [13,14]. These effects are time dependent as a result of vaccine aging and, in some cases, with significant magnitude [15], but it is not clear if they are reversible or irreversible and if there is a detrimental effect on immunogenicity. To have an accurate answer it’s necessary the integration of data from different and orthogonal techniques. A setback for assessing vaccine stability is the fact that many vaccines possess a specific biological activity that cannot be fully characterized by physico-chemical methods alone. Biological assays play an important role in the quality control of vaccine and are essential parameters of vaccine quality [16]. Ideally, a stability-indicating parameter should reflect the link between vaccine quality and efficacy, as demonstrated by immunogenicity and subsequent protective effect [17]. The aim of our study is to understand if the adsorption of antigens on AH
https://doi.org/10.1016/j.vaccine.2020.02.001 0264-410X/Ó 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: A. Colaprico, S. Senesi, F. Ferlicca et al., Adsorption onto aluminum hydroxide adjuvant protects antigens from degradation, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.001
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induces antigen protection or destabilization. We chose to focus our analysis on three antigens of the Meningococcal Serogroup B (4CMenB) vaccine [18] as a model for protein based vaccines, with the purpose to extend the method of analysis to different vaccines. We first analyzed by nano DSC antigens adsorbed (formulated antigens) and not adsorbed (unformulated antigens) on AH to compare their thermal stability. We then performed a forced degradation study on 4CMenB antigens both formulated and unformulated with AH to verify the role of the adjuvant on antigens stability and functionality. It’s difficult to characterize with antibodies the structure and stability of proteins when they are adsorbed onto the surface of aluminum salts due to steric hindrance of AH aggregates [19]. In our study we utilized a desorption method which enabled the analysis of antigens desorbed from AH in their folded form, as suggested by the SE-UPLC (SEC) analysis. This allowed the introduction of SEC as a tool to analyze desorbed antigens and of capture ELISA assay which, based on bactericidal polyclonal antibodies, could be a link between in vitro and in vivo assays. We identified important differences between the antigens thermally stressed after adsorption on AH and antigens stressed in absence of AH. We found that AH has an important protective effect on 4CMenB vaccine antigens, increases their Melting Temperature and assures their in vivo functionality also when stressed at the high temperatures tested. 2. Materials and methods 2.1. Antigens description Clinical grade material of the 4CMenB vaccine recombinant proteins was obtained from GSK Vaccines in Siena (Italy). Factor H binding protein (fHbp) fused with GNA2091, the antigens Neisseria adhesin A (NadA), the Neisseria Heparin-Binding antigen (NHBA) fused with GNA1030, were used for the study. FHbp is a surface exposed lipoprotein which induces high levels of bactericidal antibodies also protective in vivo, following passive immunization [20,21]. To enhance immunogenicity, it is fused with an accessory protein Genome derived Neisseria Antigen (GNA) 2091, another antigen identified by reverse vaccinology. NadA induces a strong bactericidal response, and is protective by passive immunization in the infant rat model [22,23]. NHBA is a lipoprotein that binds heparin, induces bactericidal antibodies in mice and is protective by passive immunization in infant rat model [24,25]. In the vaccine it is fused with an accessory protein GNA1030. The theoretical molecular weights confirmed by experimental mass spectrometry were 35 kDa for NadA, 68 kDa for NHBA-GNA1030 and 46 for fHbp-GNA2091, while isoelectric points (pI) were theoretically calculated as 4.6, 5.1, and 9.0, respectively. 2.2. Nano Differential Scanning Calorimeter (nano DSC) FHbp-GNA2091, NadA, and NHBA-GNA1030 were loaded separately in a TA instrument nano Differential Scanning Calorimeter (nano DSC) at a concentration of 0.1 mg/ml in formulation buffer (10 mM Histidine pH 6.3, 6.25 mg/ml NaCl, 2% sucrose) and when adsorbed on AH (10 mM Histidine pH 6.3, 6.25 mg/ml NaCl, 2% sucrose, 3 mg/ml AH) to monitor the differences in the amount of heat absorbed by antigens and an equal amount of matching buffer. The buffer baseline was first obtained to establish the linearity, then each antigen was scanned. An alternation of 10 cooling and heating cycles were performed from a starting temperature of 10 °C to 100 °C and 10 °C to 120 °C for NHBA-GNA1030, with a 1 °C/ s scan rate. To prevent bubble formation, cells were pressurized at a constant pressure of 3 atmospheres. The differences in heat adsorption or release between the reference cell which contains
the protein buffer and the cell of the sample, give us indication of the Melting Temperature (Tm), which is the temperature at which the 50% of protein is unfolded. 2.3. Antigen formulation AH suspension used for this study was produced at GSK Vaccines (Marburg, Germany) and also used in licensed vaccine. The formulated antigens were prepared by adsorbing the three recombinant proteins at a concentration of 0.1 mg/ml on AH (3 mg/ml) in 10 mM Histidine buffer (pH 6.3) and 2% sucrose. The final osmolality was adjusted to 300 ± 60 mOsm/kg using 2 M NaCl solution. The unformulated antigens were prepared by adding the three recombinant proteins at a concentration of 0.1 mg/ml in 10 mM Histidine buffer (pH 6.3) and 2% sucrose. The final osmolality was adjusted to 300 ± 60 mOsm/kg using 2 M NaCl solution. For the nano DSC analysis and for the in vivo study, the antigens were formulated individually. The formulations were stirred for 15 min at room temperature (RT), stored overnight (17 h) at 4 °C and analyzed the day after. The entire panel of analysis was repeated three times. 2.4. Thermal stress The formulated antigens and the unformulated antigens were split into 5 doses: one dose was not stressed (NS), the other ones were stored 17 h at 37 °C or incubated for 3 h at 50 °C, 65 °C and 80 °C. After the stress the samples were cooled to RT and immediately processed for the analysis. 2.5. Antigens recovery from Aluminum Hydroxide The antigens recovery consisted on the desorption of antigens from AH upon exposure to high concentration of phosphate buffer. Formulated antigens were mixed to a double volume of desorbing buffer (450 mM KH2PO4 pH 6.5, 15% Ethylene glycol, 0.02% Tween 80) in a 2 ml Eppendorf Protein LoBind Tube and incubated with gentle rocking agitation at 25 °C for 17 h. After incubation, the tubes were centrifuged at 2100g for 20 min. The supernatants were then removed and analyzed by Capture ELISA, RP-UPLC, SE-UPLC and SDS-PAGE. The unformulated antigens were treated with the same desorption procedure. 2.6. Reverse-Phase Ultra Performance Liquid Chromatography (RPUPLC) After the stress and the desorption procedure, formulated antigens and unformulated antigens were analyzed for protein content using an Acquity UPLC Waters chromatographic system equipped with a photodiode array detector (PDA, Waters). The samples obtained after the desorption procedure (10 ll), with a theoretical concentration of 33 mg/ml, were loaded on a C4 BEH300 column 1.7 mm (2.1 mm ID 150 mm, Waters) and antigens were eluted with a conventional RP-UPLC system utilizing a mobile phase of water/0.1% TFA and acetonitrile/0.1% TFA at a flow rate of 0.6 ml/ min. A standard curve (200–6.25 mg/ml) was prepared by twofold serial dilution of a stock of 200 mg/ml of three 4CMenB antigens in the formulation buffer. To monitor potential effects of the elution method on the antigens, a sample with the same formulation composition was treated with desorbing buffer in the same manner of formulated and unformulated antigens and used as reference control. The samples were loaded onto column and eluted with a gradient of acetonitrile (20–80%) and quantified by peak area calculation at 214 nm wavelength. Standards were analyzed both at the beginning and at the end of each analytical session.
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2.7. Size Exclusion Ultra Pressure Liquid Chromatography (SE-UPLC)
2.11. Antigen-Specific Capture ELISA
SE-UPLC is of extreme value for the analysis of multimeric antigen to monitor their quaternary structure and to probe the presence of aggregated protein in general. An Acquity UPLC H-class chromatographic system (Waters) equipped with a PDA detector was used for SEC separation with UV detection and spectral analysis. A BEH 200 Å column (4.6 mm ID 300 mm, 1.7 mm, Waters), at 30 °C was used, with the corresponding pre-column. The samples obtained after the desorption procedure (10 ll) were loaded onto the gel filtration column and eluted with an isocratic mobile phase: 0.5 M KH2PO4 pH 7.2 with 20% ACN, flow rate of 0.3 ml/min. To monitor the quality of the antigen, the UV absorbance at 214 nm were recorded and the chromatograms peaks were compared with each relative reference control and standard.
The reagents of the Meningococcal Antigen Typing System (MATS) [26] were used to assess the integrity of the antigens after the thermal stress. Briefly, after the desorption procedure, formulated and unformulated antigens were diluted in dilution buffer (0.1% BSA in PBS1X) to reach a final concentration of 10 ng/ml. Two-fold serial dilutions were plated in duplicate in ELISA plates, which were pre-coated with rabbit polyclonal antibodies against fHbp, NHBA, or NadA to capture the antigens. Plates were sealed and incubated for 1 h at 37 °C and then washed with PBS/0.05% Tween. After adding 100 ll of biotinylated rabbit polyclonal antibody, the plates were incubated for 1 h at 37 °C. Following the removal and wash of the well, 100 ll of streptavidin-HRP was added and incubated for 30 min at 37 °C. The wells were developed with o-Phenylenediamine dihydrochloride (OPD, Sigma) for 20 min at RT, the reaction was stopped by addition of 50 ll of 4 N H2SO4 and read immediately at 492 nm with an ELX 808 Biotek plate reader. A standard curve created by 2-fold dilution of a 10 ng/ml antigen stock was included on each plate. As internal quality control and to monitor the inter-assay variability, antigen at a fixed concentration (2.5 ng/ml) was plated.
2.8. SDS-PAGE Reducing SDS-PAGE was performed using NuPAGE pre-cast 4– 12% Bis-Tris Gels (Invitrogen). Aliquots of desorbed antigens (20 ll) were mixed with 4X Loading Sample Buffer (LSB) (SDS 8%, Glycerol 25%, Bromophenol blue 0.25%, DTT 6%, Tris-HCl 0.25 M pH 6.8), heated at 90 °C for 10 min and loaded into the gel. To evaluate the amount of antigens not desorbed from AH, the pellet obtained after the desorption procedure was resuspended with 0.6 M KH2PO4, 5% SDS and treated 10 min at 100 °C. The samples were centrifuged 20 min at 2100g, 20 ll of the obtained supernatant were loaded in the gel. To evaluate the amount of antigens not adsorbed on AH at the different stress temperatures, 200 ll of stressed formulations were centrifuged at 2100g for 20 min and the supernatants obtained were treated with DOC 0.05% and TCA 4.8%. After 10 min were centrifuged at 2100g for 20 min and the pellets were resuspended with 10 ll of TrisHCl pH 8 and 10 ll of LSB, heated at 90 °C for 10 min and loaded into the gel. The molecular weight standard (Seeblue plusInvitrogen) was loaded on each gel for reference. The proteins were electrophoretically separated using the Invitrogen Xcell SureLockTm Mini-Cell system (Invitrogen), stained with Easyblue Invitrogen and de-stained with milliQ Water. Gel images were then digitalized on GelDoc Biorad. 2.9. Ethical statement All animal studies were carried out in compliance with Italian legislation in force at that time, on the care and use of animals in experimentation (Legislative Decree 64/2006-A) and with Novartis Animal Welfare Policy and Standards. Protocols were approved by the Italian Ministry of Health (authorization AEC/2011–01) and by local Novartis Animal Welfare Body 2.10. Mice immunization Groups of 8 CD1 six-weeks old female mice were immunized by the intraperitoneal route using different doses (20 and 1 lg in 200 ll) of not stressed and stressed formulated antigens. Following the 3Rs recommendations, the unformulated antigens were not analyzed in vivo because we know from undisclosed data that, in absence of AH the SBA titers are lower and not comparable with the ones obtained in presence of AH, moreover, adsorbing on AH the unformulated antigens after the thermal stress could change their structure leading to misinterpret the results. For the same reason NHBA-GNA1030 and fHbp-GNA2091 stressed at 65 °C were not analyzed in vivo because, based on nano DSC data, we didn’t expect variations in SBA titers in the samples stressed at this temperature. Animals were immunized on days 1, 21 and 35 and the sera were collected at day 49.
2.12. Serum bactericidal antibody assay Serum bactericidal activity against N. meningitidis strains was evaluated as previously described [27] with pooled baby rabbit serum (Cederlane Laboratories). Briefly, bacteria were grown in MH (Muller Hinton) broth plus 0.25% glucose for approximately 1.5 h at 37 °C with shaking until early log phase (OD600) and then diluted in Dulbecco’s buffer (SIGMA) plus 1% BSA and 0.1% Glucose (DPBS) to approximately 104–105 CFU/ml. Serum bactericidal titers were defined as the serum dilution resulting in a 50% decrease in the CFU/ml after 60 min of incubation of bacteria with the reaction mixture, compared to the control. 3. Results 3.1. Nano DSC analysis of 4CMenB antigens Melting Temperatures Calorimetry is an unsurpassed tool for measuring the thermal properties of materials to establish a connection between temperature and specific physical properties of substances [28]. It is the ideal technique to study protein stability and the reversibility of thermal processes. It was also used to determine thermogram profile before and after desorption of antigen from AH, to asses impact of desorption process on antigen stability [29]. Parameters like the shape of the thermogram and the Tm provide the most immediate information on increased or decreased stability of the protein and can be used in pre-formulation work to scout the best formulation conditions to improve protein stability. The three 4CMenB vaccine antigens were analyzed in nano DSC in order to identify their Tm when formulated or unformulated with AH. The analysis of antigens formulated with AH showed a trend of increased Tm respect to unformulated antigens, as shown in Table 1, with the notable exception of the accessory protein GNA2091 fused with fHbp. The DSC thermogram of NadA changes dramatically upon adsorption onto AH, we in fact measured a Tm that was found to be 10 °C higher with respect to the Tm that was measured in absence of AH. Also fHbp and NHBA-GNA1030 were affected by adsorption onto the AH matrix which caused an increase of about 2 °C and 5 °C respectively, thus confirming that AH adsorption appears to stabilize these antigens. With the aim to observe antigen modifications and investigate if they correlate with alteration in bactericidal titers, we chose to stress the three proteins at the temperatures
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Table 1 Melting temperature comparison of formulated and unformulated 4CMenB antigens. The fHbp-GNA2091 presented three peaks each belonging to a component of the fusion protein. Antigens
Portion or Domain providing signal
Tm of Unformulated Antigens
NHBA-GNA1030 fHbp-GNA2091
NHBA GNA2091 fHbp N-Term fHbp C-Term Full lenght
98 59 67 82 50
NadA
°C °C °C °C °C
Tm of Formulated Antigens 103 °C 50 °C 69 °C 84 °C 60 °C
they unfold. Based on nano DSC data we therefore decided to stress the 4CMenB antigens (i.e. NadA, NHBA-GNA1030, fHbp-GNA2091), formulated and unformulated with AH at three different stress conditions, namely 17 h at 37 °C, 3 h at 50 °C which corresponds to the Tm of unformulated NadA, 3 h at 65 °C, temperature at which, according to the Tm, formulated NadA should be denatured, and 3 h at 80 °C, which is a temperature at which we expect to find NadA totally degraded, fHbp-GNA2091 partial functional and NHBA-GNA1030 still totally functional. 3.2. RP-UPLC: Quantification of stressed unformulated and formulated antigens RP-UPLC resolves proteins with different hydrophobic properties that may have undergone significant primary structure changes. When antigens were stressed in absence of AH we observed a lower recovery after 3 h at 80 °C, probably due to precipitation (Fig. 1), while for NadA the recovery start to decrease after 3 h at 50 °C, in correspondence to its Tm. We observed that antigens recovery from AH was stress-temperature dependent, in fact, while the not stressed antigens were totally recovered from AH (Fig. 1), the desorption rate slowly decreased with increasing the stress temperature. This effect could be due to a rearrangement of antigens onto AH surface which interfere with desorption [4,30]. This hypothesis was confirmed when we analyzed the same desorbed samples by SDS-PAGE which showed that, the higher the stress temperature is, the more difficult to desorb antigens from AH (Fig. 2a). We also analyzed the residual formulation pellet obtained after 17 h of desorption, treated with harsher condition and we confirmed that, increasing the temperature, an increasing quantity of antigens remain adsorbed on AH (Fig. 2b). When analyzed 24 h after the preparation, in the formulation supernatants is usually present the fHbp-GNA2091 in a quantity <10% of the total amount and it adsorbs more strongly onto AH after storage at 4 °C. In the analysis of stressed formulations we found that increasing the temperature, to a lower recovery of antigen corresponds also a lower amount of antigen in the formulation supernatant (Fig. 2c), suggesting that it adsorbs more strongly onto AH surface, as hypothesized with RP-UPLC results. 3.3. Capture ELISA analysis of stressed unformulated and formulated antigens The ideal condition to analyze antigens alteration in a vaccine would be when still adsorbed on adjuvant. AH characteristics, such as its greater dimension respect to antigens [19] which create a steric interference to antibody accessibility, the fact that it could mask important epitopes for antibodies binding, and a possible antibodies adsorption on its surface if not well saturated, induced us to abandon the idea of a direct detection of antigens on AH, we in fact obtained variable results and we directed our investigation on antigens desorbed from AH after stress. For this reason, we
introduced a desorption procedure which didn’t alter aggregation status of proteins (see Fig. 5, not stressed samples). After being stressed on AH, desorbed antigens were analyzed also in capture ELISA to measure their capacity to be captured by specific, reproducible and standardized polyclonal antibodies coated on plates. The analysis with a biological assay based on the use of bactericidal antibodies, helped us to understand if the qualitative changes observed in SE-UPLC (Fig. 5) were so significative to alter antigen functionality. We obtained a good recovery of not stressed antigens (Fig. 3) which drastically decreased after 3 h of storage at 80 °C, when the three antigens were less recognized by antibodies (NHBA-GNA1030: 20%, fHbp-GNA2091: 3%, NadA: 4%). Differently, antibody recognition of formulated NadA starts to decrease already after 3 h of storage at 50 °C (recovery 45%) and 65 °C (recovery 10%) (Fig. 3). The analysis of unformulated antigens had the same trend of the formulated ones, except for NadA, for which we observed a complete loss of antibody recognition after 3 h at 50 °C in absence of AH, as expected based on nano DSC analysis, which indicated that the Tm of unformulated NadA was 50 °C (Table 1). This support the thesis that AH protects antigen from thermal degradation.
3.4. Ratio capture ELISA/RP-UPLC Because of we found that when the formulations were stressed at high temperatures we couldn’t totally desorb antigens from AH (Figs. 1–2), and since we analyzed in capture ELISA desorbed antigens, to avoid an underestimation of antigen functionality when stressed at higher temperatures, we considered the percentage of recovery obtained in RP-UPLC as the maximum amount of antigen we can desorb, and impose it as the 100% with which compare the recovery obtained in capture ELISA (Fig. 4). Interestingly we found that, even if NHBA-GNA1030 at 80 °C was less desorbed from AH, due probably to a rearrangement of antigen on adjuvant, it was still totally recognized by bactericidal antibody, which induced us to speculate that also in vivo the stressed formulation could still induce protection. When formulated fHbp-GNA2091 was stressed for 3 h at 65 °C, almost the totality of the desorbed antigen was still recognized by antibody. When stressed 3 h at 80 °C, only the 13% of the desorbed antigen was still captured by antibody, suggesting a decrease of functionality which we hypothesized could be found also in SBA analysis. As expected from nano DSC analysis, we observed a significative decrease in the percentage of formulated NadA still recognized by antibody after a thermal stress of 3 h at 65 °C (28%), and after being stressed 3 h at 80 °C the antigen is no more recognized by antibodies. Antibodies coated on plate are bactericidal, therefore, capture ELISA could be considered as ‘‘functional assay” for the part endowed of SBA activity and these results could predict the functionality of antigens in vivo, when stressed at these high temperatures. Unformulated antigens showed a similar trend, they differentiate from formulated antigens after 3 h at 65 °C when the protective effect of AH became more evident. For NadA we observed an important differentiation between formulated and unformulated antigen already after 3 h at 50 °C, when the unformulated antigen was no more recognized by antibody, confirming our hypothesis based on nano DSC analysis, according with which AH adsorption increases antigen Tm, in the case of NadA it increases from 50 °C in absence of AH to 60 °C when formulated with AH. These results showed that the adsorption on AH protect antigens from thermal degradation and indicated that the three vaccine antigens are very robust and resistant to high temperatures, in fact, NHBA-GNA1030 is still recognized by bactericidal antibody at 80 °C, fHbp-GNA2091 recognition decrease only at 80 °C and NadA is still intact at 50 °C. The ratio between capture ELISA and RP-UPLC provides a very useful way to understand
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NHBA-GNA1030
% of antigen recovery
120
Unformulated Antigens Formulated Antigens
90 60 30 0 Not stressed 37°C
50°C
65°C
80°C
fHbp-GNA2091
% of antigen recovery
120
Unformulated Antigens Formulated Antigens
90 60 30 0 Not stressed 37°C
50°C
65°C
80°C
NadA % of antigen recovery
120
Unformulated Antigens Formulated Antigens
90 60 30 0 Not stressed 37°C
50°C
65°C
80°C
Fig. 1. Percentage of antigen recovery in RP-UPLC at different temperatures. The recovery of unformulated antigens decreased in correspondance of antigens Tm. The desorption rate of formulated antigens decreased with increasing the stress temperature (dotted bars), probably due to a rearrangement of antigens on Alum. Average of three experiments.
how much antigen is still somehow ‘functional’ in the recovered amount. 3.5. SE-UPLC SE-UPLC separates analytes on the basis of a combination of their hydrodynamic size, diffusion coefficient, and surface properties. It can show protein polymerization, oligomerization or degradation. It was particularly useful to understand whether changes occurred on the stressed unformulated and formulated antigens in comparison with the original untreated sample. Our desorption procedure allowed us to analyze antigens desorbed from AH in their folded form. Thermally stressed unformulated and formulated antigens desorbed from AH were injected in SE-UPLC for a
qualitative analysis. We verified that the desorption treatment didn’t cause protein unfolding and didn’t impact antigen profile (the Not stressed formulated antigen has the same profile of Standard), moreover the unformulated antigens received the same desorption treatment of the formulated antigens so that all the changes we observed in SE-UPLC were assignable to thermal effect. We observed only a decrease in peaks intensity with the increasing temperature of formulated NHBA-GNA1030 and fHbp-GNA2091 (Fig. 5), which were due to the lower recovery of antigens we have previously described (Fig. 1). We could also observe an increase of an aggregate belonging to unformulated NHBA-GNA1030 (confirmed by SE-UPLC on single antigen), which became more intense with increasing temperature, while remained constant when the antigen was adsorbed on AH. There is a conformational change of
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(a)
NS
37 °C 50 °C 65 °C 80 °C
(b)
NS 37 °C 50 °C 65 °C 80 °C
(c)
NS
37 °C
50 °C 65 °C 80°C
NHBA-GNA1030
fHbp-GNA2091 NadA
Ags desorbed ON
Ags residues on AH
Ags in the Supernatant
Fig. 2. SDS-PAGE of desorbed antigens (a), antigens residues still adsorbed on AH after desorption (b) and antigens in formulation supernatant (c). SDS-PAGE confirms RPUPLC results: the higher the temperature is, the less we recover antigens from AH; they remain adsorbed on it, probably due to rearrangement phenomena.
unformulated NadA in correspondence of Tm (50 °C) in fact, increasing the temperature, there is a progressive prevalence of the monomeric form (confirmed by SE-UPLC on single antigen) respect to the trimeric form. The two forms are well discriminated in capture ELISA (Figs. 3–4), in fact the monomeric form is no more recognized by antibody. On the other side, when formulated with AH, NadA remains in its trimeric form at 50 °C and become monomer only at 65 °C. Results clearly indicated that the three antigens are stabilized by the adsorption on AH. 3.6. Serum bactericidal antibody levels (SBA) of stressed formulations SBA is the most important method for measuring functional activity of serum antibodies against meningococcus [31–35]. To evaluate immunogenicity and functionality in vivo, CD1 mice were immunized with monovalent stressed and not stressed formulations at different antigens doses. Complement-mediated serum bactericidal antibody titers was measured 14 days after the third immunization. The bactericidal activity of sera resulting from mice immunized with stressed and not stressed formulated NHBAGNA1030 was tested against two strains, M4407 and M3279, respectively highly and lowly expressing the serobase protein, as estimated by Meningococcal Antigen Typing System (MATS) [26] (Table 2A). Thermally stressed formulated NHBA-GNA1030 had the same serum bactericidal titers of not stressed formulation, in agreement with what we observed in capture ELISA/RP-UPLC ratio. Sera of mice immunized with stressed formulated fHbp-GNA2091 were tested against the strains MC58 and M01-0240660 (UK660), respectively highly expressing and lowly expressing the protein by MATS. In UK660, 1 mg of antigen induced similar SBA titers even after a stress of 3 h at 50 °C. When the stress was for 3 h at 80 °C SBA titers decreased by 3 folds, confirming what we had predicted analyzing capture ELISA/UPLC ratio (Table 2B). With 20 mg of fHbpGNA2091 we reached the plateau, so it was not possible to appreciate differences in SBA titers when the formulation was stressed at 80 °C. The serum bactericidal activity of mice immunized with formulated NadA was tested against the strains 5/99 and 2996,
respectively highly expressing and lowly expressing the protein by MATS. As expected, based on capture ELISA data, bactericidal activity decreased after a stress of 3 h at 65 °C (Table 2C), which is a temperature slightly higher than NadA Tm (60 °C) when adsorbed on AH (Tab. 1). It is very evident how the presence of AH confers stability to NadA which, when adsorbed on AH, is still functional after a stress of 3 h at 50 °C while, in absence of adjuvant, at the same temperature is unfolded (Fig. 5) and no more recognized by bactericidal antibody (Fig. 3). These results are in complete agreement with what we predicted basing on the ratio capture ELISA/RP-UPLC (Fig. 4). 4. Discussion While the role of AH as immune potentiator is well documented, its role on antigen stability is still under discussion. For this reason we analyzed three antigens of the 4CMenB vaccine both formulated and unformulated with AH to verify if the adsorption on adjuvant stabilizes or destabilizes antigens. By nano DSC analysis we found that, when adsorbed onto AH surface, the Melting Temperature increased from 2 to 10 °C, depending on antigen. We performed a forced degradation study to deliberately alter either formulated or unformulated antigens till their end-point identified by nano DSC and cause alteration in immune response measured by Serum Bactericidal Assay. We observed that, when antigens were thermally stressed adsorbed on AH, they rearranged or precipitate on its surface and at high temperature the adsorption became stronger, so that was difficult to desorb them from AH. In RP-UPLC we had a lower recovery of antigens in correspondence of higher stress temperature, and in SE-UPLC we observed a peak intensity decrease. The analysis of desorbed antigens in capture ELISA indicated that the three 4CMenB vaccine antigens were very robust and stable, in fact, only elevated temperature of stress induced decrease of antibodies recognition. Because of antibodies coated on capture ELISA plates were bactericidal, we hypothesized that we could obtain the same results in vivo, in fact, the analysis of sera bactericidal activity measured by SBA, totally confirmed the results of capture ELISA. On the other side, when antigens were
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NHBA-GNA1030
% of antigen recovery
120
Unformulated Antigens Formulated Antigens
90 60 30 0 Not stressed
37 °C
50 °C
65 °C
80 °C
fHbp-GNA2091
% of antigen recovery
120
Unformulated Antigens Formulated Antigens
90 60 30 0 Not stressed 37 °C
50 °C
65 °C
80 °C
NadA
% of antigen recovery
120
Unformulated Antigens Formulated Antigens
90 60 30 0 Not stressed
37 °C
50 °C
65 °C
80 °C
Fig. 3. Percentage of antigens recovery in capture ELISA plates. Comparison between stressed formulated antigens (dotted bars) and stressed unformulated antigens (black bar). There is a great impact of AH on NadA stability: after 3 h at 50 °C the unformulated NadA is no more recognized by antibodies, while when formulated with AH loses its recognition only after 3 h at 65 °C, as predicted by nano DSC analysis. Average of three experiments.
stressed in absence of AH, were more sensible to thermal stress, in SE-UPLC we observed an increase of an aggregate belonging to NHBA-GNA1030 which became more intense when temperature increased. In absence of AH and in correspondence of its Tm NadA became a monomer, while when stressed at the same temperature when adsorbed on AH, remained in its trimeric form which, unlike the monomer, is still recognized by bactericidal antibody. Results demonstrated that AH has an important effect of protection on the three investigated antigens, limiting the thermal degradation in correspondence of their respective Tm. The mechanisms of protection are complex and do not follow a general rule. Adsorption on a highly charged surface like that of AH has the potential to change the stability, the flexibility and the hydration shell of the
antigen. Antigen will react to adsorption onto AH by finding a new folding state dictated by the equilibrium between the external adsorption forces competing with the internal stabilizing interactions and this new equilibrium could be one key possible mechanism leading to antigen stabilization/destabilization upon AH adsorption. If the adsorption derived forces will not be able to impact the quaternary/tertiary organization of the antigen, this will likely be trapped in a state where it still retains important folding features. At the same time, the interaction with the adjuvant will also reduce the antigen accessibility to denaturing agents/stress resulting in an increased stabilization and functionality, as seen in our data. The different proteins adsorption and stability profiles explain the variable results reported in literature,
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Ratio Capture ELISA/RP
NHBA-GNA1030
Formulated Antigens
100
Unformulated antigens
80 60 40 20 0 Not stressed 37 °C
50 °C
65 °C
80 °C
Ratio Capture ELISA/RP
fHbp-GNA2091
100
Formulated Antigens Unformulated antigens
80 60 40 20 0 Not stressed 37 °C
50 °C
65 °C
80 °C
Ratio Capture ELISA/RP
NadA Formulated Antigens
100
Unformulated antigens 80 60 40 20 0 Not stressed 37 °C
50 °C
65 °C
80 °C
Fig. 4. Representation of the ratio capture ELISA/RP-UPLC. In the figure is reported the percentage of desorbed formulated antigens (solid line) and unformulated antigens (dotted line) still recognized by antibodies after thermal stress. AH has a protective effect on antigens, above all on NadA which at 50 °C is still functional when adsorbed on AH Average of three experiments.
highlighting the need to study the matter case by case. We can conclude that AH has a role not only as enhancer of antibody production or as antigen depot, it also has the potential to preserves antigens integrity, increasing their stability and protecting them from thermal degradation. Contributions All authors reviewed and approved the final version of the manuscript. AC, MU, MP: conceiving and designing the experiments. AC: performing experiments and writing the manuscript. AC, MU, MP, DOH: analyzing data and interpreting experiments results. SS: setting up the Desorption method and the SE-UPLC method.
BB and FF: setting up, performing and interpreting SBA analysis. AC, SS, FF, BB, MU, MP, DOH: critically reviewing the manuscript. Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: This work was sponsored by Novartis Vaccines and Diagnostics Srl (now part of the GSK group of companies) which was involved in all stages of the study conduct and analysis. AC, SS, FF, BB, MU, MP and DOH were employees of Novartis Vaccines and Diagnostics Srl at the time of the study (now part of the GSK group of companies). AC, SS, FF, BB and DOH are now employees of the GSK group of companies.
Please cite this article as: A. Colaprico, S. Senesi, F. Ferlicca et al., Adsorption onto aluminum hydroxide adjuvant protects antigens from degradation, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.001
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Unformulated Antigens
Formulated Antigens
0.040
0.040
NHBAGNA1030
0.030
NHBAGNA1030
0.030
NadA 0.020
0.010
0.020
0.010
0.000
0.000
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
10.50
11.00
11.50
12.00
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
Minutes
fHbpGNA2091
10.50
11.00
11.50
12.00
11.00
11.50
12.00
11.00
11.50
12.00
fHbpGNA2091
NadA
0.020
AU
AU
10.00
NHBAGNA1030
0.030
NadA
0.010
0.020
0.010
0.000
0.000
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
10.50
11.00
11.50
12.00
5.00
Minutes
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
10.50
Minutes
0.040
0.040
0.030
NadA
NHBAGNA1030
fHbpGNA2091
fHbpGNA2091
NadA
NadA monomer
0.020
NHBAGNA1030
0.030
AU
AU
9.50
0.040
NHBAGNA1030
0.030
3h 50 °C
9.00
Minutes
0.040
ON 37 °C
fHbpGNA2091
NadA AU
AU
Not stressed
fHbpGNA2091
0.010
0.020
0.010
0.000
0.000
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
10.50
11.00
11.50
12.00
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
Minutes
9.00
9.50
10.00
10.50
Minutes
0.040 0.040
NHBAGNA1030
0.030
fHbpGNA2091
NHBAGNA1030
0.030
0.020
0.020
NadA monomer
0.010
0.010
0.000
0.000
5.00
5.00
fHbpGNA2091
NadA AU
3h 65 °C
AU
NadA
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
10.50
11.00
11.50
5.50
6.00
6.50
7.00
7.50
8.00
12.00
8.50
9.00
9.50
10.00
10.50
11.00
11.50
12.00
11.00
11.50
12.00
Minutes
Minutes 0.040
0.040
fHbpGNA2091
NHBAGNA1030
0.030
0.030
NadA
0.020 AU
AU
NadA
3h 80 °C
0.010
fHbpGNA2091
NHBAGNA1030
0.020
0.010
0.000 0.000
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
10.50
11.00
11.50
12.00
5.00
5.50
6.00
6.50
7.00
7.50
8.00
Minutes
8.50
9.00
9.50
10.00
10.50
Minutes
Fig. 5. Formulated antigens (in green) and unformulated antigens (in orange) profile in SE-UPLC after thermal stress, compared to the antigens Standard (in black). The thermal degradation of formulated antigens is slower respect to the degradation of unformulated antigens. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 2 Bactericidal titers elicited in mice by stressed and not stressed (N.S.) formulated antigens against antigen-specific strains. In (A) is shown SBA titers against NHBA-GNA1030. In both strains and for both doses tested there were no differences between stressed and not stressed vaccine. In (B) we observed a decrease of SBA titers against fHbp-GNA2091 in UK660 strain, when the formulation with 1 mg of antigen was stressed 3 h at 80 °C. In (C) is visible an important decrease of SBA titers against NadA after 3 h at 65–80 °C. All these results are comparable to the ratio capture ELISA/RP-UPLC (Fig. 4). (A) NHBA-GNA1030 Tested strain
M4407
Tested strain
M3279
Stress conditions
N.S.
37 °C
50 °C
80 °C
Stress conditions
N.S.
37 °C
50 °C
80 °C
SBA titers with 20 mg Ag SBA titers with 1 mg Ag
16384 16384
16384 2048
16384 16384
16384 16384
SBA titers with 20 mg Ag SBA titers with 1 mg Ag
1024 2048
4096 512
2048 4096
1024 2048
(B) fHbp-GNA2091 Tested strain
MC58
Tested strain
M01 0240660/UK660
Stress conditions
N.S.
37 °C
50 °C
80 °C
Stress conditions
N.S.
37 °C
50 °C
80 °C
SBA titers with 20 mg Ag SBA titers with 1 mg Ag (C) NadA
65536 65536
65536 65536
65536 65536
16384 16384
SBA titers with 20 mg Ag SBA titers with 1 mg Ag
16384 16384
8192 16384
16384 8192
8192 1024
Tested strain
5/99
Tested strain
2996
Stress conditions
N.S.
37 °C
50 °C
65 °C
80 °C
Stress conditions
N.S.
37 °C
50 °C
65 °C
80 °C
SBA titers with 20 mg Ag SBA titers with 1 mg Ag
16384 16384
16384 16384
16384 32768
256 512
256 256
SBA titers with 20 mg Ag SBA titers with 1 mg Ag
1024 256
2048 512
2048 256
128 256
256 128
Please cite this article as: A. Colaprico, S. Senesi, F. Ferlicca et al., Adsorption onto aluminum hydroxide adjuvant protects antigens from degradation, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.001
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Please cite this article as: A. Colaprico, S. Senesi, F. Ferlicca et al., Adsorption onto aluminum hydroxide adjuvant protects antigens from degradation, Vaccine, https://doi.org/10.1016/j.vaccine.2020.02.001