ABSTRACT The heterogeneous composition of vaccine formulations and the relatively low concentration make the characterization of the protein antigens extremely challenging.

Aluminum-containing adjuvants have been used to enhance the immune response of several antigens over the last 90 years and still remain the most commonly used. Here, we show that solid-state

NMR and isotope labeling methods can be used to characterize the structural features of the protein antigen component of vaccines and to investigate the preservation of the folding state of

proteins adsorbed on Alum hydroxide matrix, providing the way to identify the regions of the protein that are mainly affected by the presence of the inorganic matrix. l-Asparaginase from

NANOROD FOR ELUCIDATING THE ANTIGEN ADSORPTION BEHAVIOR Article Open access 23 June 2022 FORMALDEHYDE TREATMENT OF PROTEINS ENHANCES PROTEOLYTIC DEGRADATION BY THE ENDO-LYSOSOMAL PROTEASE

CATHEPSIN S Article Open access 14 July 2020 ENSILICATED TETANUS ANTIGEN RETAINS IMMUNOGENICITY: _IN VIVO_ STUDY AND TIME-RESOLVED SAXS CHARACTERIZATION Article Open access 08 June 2020

INTRODUCTION Vaccination is one of the major contributors to the control of infections in human population globally. Insoluble aluminum salts have been used to enhance the immunogenicity of

antigens against bacterial and viral infections since 1926, when Glenny et al. reported their use to improve the response of diphteria toxoid.1 Recently other adjuvants based on oil-in-water

emulsions (i.e. MF59, AS03) and liposomes have been used for licensed vaccines and other candidates at different stages of research and development.2,3 However, the aluminum salts are the

most commonly used adjuvants for commercial vaccines and, also due to their long-term success, they still remain the “gold standard” against a new adjuvant.4 For licensed vaccines,

aluminum(III) hydroxide (AlumOH) and aluminum(III) phosphate (AlumP) are the most commonly used adjuvants. AlumOH is a chemically crystalline aluminum(III) oxyhydroxide (AlOOH), prepared by

exposing soluble aluminum(III) salts (generally Al(H2O)6Cl3 or AlK(SO4)2) to alkaline conditions to obtain a suspension which is finally dehydrated under hydrothermal conditions. AlumP is a

noncrystalline compound generated by incorporation of phosphate which interferes with the crystallization process. AlumP can be prepared by mixing the aluminum salt Al(H2O)6Cl3 or AlK(SO4)2

with a basic solution of trisodium phosphate, or directly mixing aluminum salt with phosphate solution, followed by precipitation with sodium hydroxide. The substitution of hydroxyl groups

of AlumOH with phosphate groups results in the formation of aluminum hydroxyphosphate, Al(OH)x(PO4)y, a nonstoichiometric compound in which the ratio of hydroxyls to phosphate depends on the

precipitation conditions.4 In the final formulation the antigen is adsorbed on the Alum-based adjuvant and administered as precipitate. After the injection, a fraction of the antigen is

released in the extracellular fluid and cleared from the injection site. These adjuvants enhance the immune response by a slow release of the antigen from the injection site and, more

important, through activation of the dendritic cells and stimulation of CD4+ T cells.5 However, probably due to the complexity and several immunological pathways operating simultaneously,

the mechanism of action of aluminum adjuvants for enhancing the immune response remains not fully understood, although they have been used over many years in vaccines for human use.

Electrostatic interaction, phosphate ligand exchange, hydrogen bonding and van der Waals interactions may be involved in the adsorption mechanism depending on antigen, adjuvant, excipients,

pH and ionic strength.6 However, the interaction with the adjuvant may alter folding, conformation and stability of the antigen.6 For folded protein antigens, electrostatic forces are

reported to dominate the interaction with the AlumOH hydrated gel in a manner dependent on the pH and isoelectric point.7 Alteration of folding and native conformational state of the

epitopes may affect the immune response by influencing the antigen processing and presentation, the amount of protein bound to the adjuvant and its binding affinity.8 Also the long-term

stability of the antigen in the final formulation is extremely important for the effectiveness and commercial viability of the vaccine. Therefore, the characterization of the protein antigen

bound to the aluminum gel adjuvant is particularly relevant for the development of more effective and stable vaccine. Unfortunately, the heterogeneous composition of vaccines has hampered

for long time the biophysical and structural characterization of the protein component within the formulation. Desorption from the aluminum adjuvant and elution have often been used to

analyze post-formulation antigens. However, this strategy does not provide structural information on the antigen when adsorbed on the adjuvant. Attenuated total reflectance

Fourier-transformed infrared and fluorescence spectroscopy, circular dichroism and differential scanning calorimetry have been used to investigate adjuvant-interacting protein antigens.9,10

More recently, electron microscopy has been used to characterize the antigens in adjuvant bound states.11 However, this approach is not for general use, and it may be feasible only with

extremely large proteins or protein assemblies. In the last years, solid-state NMR is emerging as an outstanding spectroscopic technique in structural

biology,12,13,14,15,16,17,18,19,20,21,22,23,24,25 and to investigate, at atomic detail, difficult protein systems in many different states, including biosilica-entrapped proteins,

hydroxyapatite-protein composites, PEGylated and polysaccharide-conjugated proteins and proteins grafted onto nanoparticles.26,27,28,29,30,31,32,33 In particular, soluble proteins and

protein assemblies,34 membrane proteins, such as bacterial porins17 and transmembrane helix proteins,35 viral capsid components36 and RNA37 have been characterized by solid-state NMR

spectroscopy, opening up promising opportunities for the study of different classes of antigens. Here we show that atomic structural details on protein antigens adsorbed on Aluminum gel

adjuvant can be achieved by solid-state NMR from vaccine formulations obtained starting from isotopically enriched antigens and stored for several months. The following NMR analysis

performed on _Escherichia coli_ l-Asparaginase (ANSII) sheds light (i) on the folding state of the protein bound to aluminum adjuvant, (ii) on the protein regions involved in the interaction

with gel, and (iii) it provides a new tool for vaccines formulation development and stability studies. RESULTS NMR SPECTROSCOPY AlumOH adjuvanted formulation of uniformly isotopically

enriched ANSII [U-13C-15N] was used as vaccine model and investigated by solid-state NMR. Despite the low amount of protein absorbed on the inorganic matrix, the 2D amide-carbon alpha (2D

15N 13C NCA) and amide-carbonyl (2D 15N 13C NCO) correlation spectra of ANSII-AlumOH (Fig. 1a, b, respectively), collected on sedimented material obtained by centrifugation, are of high

quality, and comparable for the number of cross-peaks detected to the 2D 15N 13C NCA and 2D 15N 13C NCO spectra collected on rehydrated freeze-dried ANSII. In both N−C correlation spectra

the resonances are superimposable to those of the rehydrated freeze-dried ANSII (Fig. 1), immediately demonstrating that the three-dimensional structure of the protein is preserved after

adsorption on the inorganic salt. The assignment of the 2D 15N 13C NCA spectrum of ANSII-AlumOH (see Supplementary Table 1) was easily obtained by comparison with the 2D 15N 13C NCA

collected on the rehydrated freeze-dried ANSII, and also using the information from the 2D 13C-13C correlation spectrum (dipolar assisted rotational resonance, DARR) acquired on

ANSII-AlumOH. DATA ANALYSIS The assignment of the spin systems allowed the analysis of the chemical shift perturbation (CSP). The CSP data reveal that the residues experiencing the largest

changes are located on the protein surface with negative electrostatic potential (Fig. 2). In particular, the largest CSP are observed for residues in the region between Ala-250 and Lys-310,

which possesses a wide distribution of negative charge. The presence of electrostatic interactions between the negatively charged surface of the protein and the positive surface of the

inorganic material is further supported by the properties of the aluminum oxyhydroxide that has an isoelectric point of about 11.4, and at pH 7.5 exhibits a positive surface charge.38 These

findings are consistent with the mechanism proposed for the adsorption of folded antigen proteins onto the AlumOH. DISCUSSION The application of structural biology to develop new vaccines

has already proved its effectiveness.39 Engineered antigens incorporating protective determinants have been developed by NMR-based structural methodologies to improve protection, safety and

industrial scale production.40 Recently, we have demonstrated that high-resolution SSNMR, based on 13C detection, can be applied to assess the preservation of the folding of

silica-encapsulated enzymes,27 and track the chemical shift perturbation on the protein surface induced by the interaction with ligands.41 ANSII from _E. coli_ is a homotetrameric assembly

of 138 kDa with D2 symmetry. This protein is in clinical use since 1967 against childhood acute lymphoblastic leukemia but has now been largely replaced by its PEGylated form, which exhibits

longer‐lasting activity and, more important, a lower immunogenicity.42 The previously reported characterization of native ANSII and its antigenic properties make it a suitable model to

investigate the potential of new NMR methods for vaccines development and characterization. The amount of protein antigen adsorbed to the adjuvant and the heterogeneity of the vaccine

formulation are two important limiting factors for their biophysical characterization. However, these limitations can be overcome today by using the high sensitivity of solid-state NMR

combined with isotopic labeling methodologies. Therefore, the use of solid-state NMR and structural methodologies to characterize adsorbed antigens promises to solve several challenges

frequently encountered in vaccine development. For example, the destabilization of protein antigen structure upon the adsorption to the aluminum adjuvants has been suggested to play a role

in immune stimulation. In particular, protein unfolding may favor the proteolytic degradation of the antigen and the presentation of the fragments to the immune cells.6 At the same time, for

some vaccines, other studies have shown that the loss of the native secondary and tertiary structure can result in partial loss of immunogenicity.43,44 In this respect, the information

provided by solid-state NMR on folding preservation could be decisive to determine the molecular basis of the loss of efficacy and to design vaccine with an improved immunogenicity. A

further potential application of the solid-state NMR to vaccine development concerns the optimization of the experimental conditions for the adsorption of antigen to aluminum salts. Usually,

the adsorption of the antigen protein is optimized by changing the pH and buffer components that directly affect the electrostatic interactions (i.e. zeta potential). However, the amount of

protein antigen adsorbed to the aluminum salt is determined by measuring the concentration of the residual free protein in solution without any quantitative and qualitative information on

the protein bound to the adjuvant. In this respect, the collection of mono- and multidimensional solid-state NMR spectra on formulations containing isotopically enriched protein antigens

allows for a completely new approach providing semiquantitative information on the adsorbed protein antigen and on its folding state, suitable for driving structure-based optimization of

vaccine formulation and manufacturing process. Also accelerated stability studies and mechanistic studies to investigate the exposure to high temperatures, freeze-thaw events and low pH

would benefit from the use of this new methodology. Moreover, it should be pointed out that this methodology can be applied to a wide range of proteins because the labeling of antigens in

eukaryotic expression systems, although highly expensive for academia, is nowadays feasible.45,46,47,48,49,50 Finally, the extension of the cryo-probe technology to solid-state NMR and the

forthcoming increase in magnetic field strength of the NMR instruments are expected to improve further the sensitivity of the experiments allowing a more detailed characterization, or

decreasing the amount of protein required for the detection. METHODS EXPRESSION AND PURIFICATION OF UNIFORMLY ISOTOPICALLY ENRICHED ANSII [U-13C-15N] _Escherichia coli_ C41(DE3) cells were

transformed with pET-21a(+) plasmid encoding ANSII gene. The cells were cultured in 13C, 15N-enriched minimal medium (M9) containing 0.1 mg/mL of ampicillin, grown at 310 K, until OD600

reached 0.6–0.8, then induced with 1 mM isopropyl _β_-d-1-thiogalactopyranoside. They were further grown at 310 K overnight and then harvested by centrifugation at 6500 rpm (JA-10 Beckman

Coulter) for 15 min at 277 K. The pellet was suspended in 10 mM Tris-HCl, pH 8.0, 15 mM EDTA, 20% sucrose buffer (60 mL per liter of culture) and incubated at 277 K for 20 min upon magnetic

stirring. The suspension was centrifuged at 10,000 rpm (F15-6x100y Thermo Scientific) for 30 min and the supernatant discarded. The recovered pellet was re-suspended in H2O milli-Q (60 mL

per liter of culture) and newly incubated at 277 K for 20 min under magnetic stirring. Again the suspension was centrifuged at 10,000 rpm (F15-6x100y Thermo Scientific) for 30 min. The

pellet was discarded, whereas the supernatant was treated with ammonium sulfate. Still under magnetic stirring, solid ammonium sulfate was added in aliquots up to 50% saturation. The

precipitate was removed by centrifugation, then further ammonium sulfate was added up to 90% saturation to trigger the precipitation of ANSII, which was recovered again by centrifugation.

The precipitated ANSII was re-dissolved in a minimal amount of 20 mM Tris-HCl buffer at pH 8.6 and dialyzed extensively against the same buffer. ANSII was purified by anionic-exchange

chromatography using a HiPrep Q FF 16/10 column (GE Healthcare Life Science). The protein was eluted in 20 mM Tris-HCl buffer at pH 8.6 with a linear 0–1 M NaCl gradient. Fractions

containing pure ANSII were identified by Coomassie staining SDS-PAGE gels, then joined and dialyzed extensively against 50 mM phosphate buffer at pH 7.5. PREPARATION OF VACCINE FORMULATION

Commercially available AlumOH (Sigma) was used. To reduce the phosphate content and assure a complete adsorption to AlumOH adjuvant, 10 mL of ANSII [U-13C-15N] protein at the concentration

of 0.583 mg/mL in 150 mM sodium phosphate (pH 7.5) was dialyzed and concentrated to 2 mL volume by using a Vivaspin 5 kDa molecular weight cutoff membrane (Sartorius). A protein

concentration of 2.77 mg/mL was obtained as estimated by MicroBCA commercial kit (Thermo). Fifty milliliters of AlumOH adjuvanted formulation at a protein concentration of 100 µg/mL (with 2 

mg/mL of Al(OH)3 and 9 mg/mL of NaCl) was prepared by mixing 1.805 mL of ANSII [U-13C-15N] (5 mg totally), 23.195 mL MilliQ H2O, 25 mL AlumOH at 4 mg/mL Alum(OH)3 and 18 mg/mL NaCl. To

estimate the amount of protein adsorbed to the AlumOH adjuvant, the hydrogel was pelleted at 15,000 rpm for 1 min and the protein content estimated on the supernatant. SAMPLE PREPARATION AND

NMR MEASUREMENTS SSNMR experiments were recorded on a Bruker Avance III spectrometer operating at 800 MHz (19 T, 201.2 MHz 13C Larmor frequency) equipped with Bruker 3.2 mm Efree NCH

probe-head. All spectra were recorded at 14 kHz MAS frequency and the sample temperature was kept at ~290 K. The sample of ANSII-AlumOH was stored for 6 months at 277 K to reproduce a

possible shortest shelf-life of a commercial vaccine. A mild centrifugation was used to separate the colloidal AlumOH adjuvant with the adsorbed ANSII from the supernatant. The hydrogel was

pelleted at 10,000 rpm for 1 h at 4 °C using an ALC multispeed refrigerated PK121R centrifuge (rotor model A-M10). Then, the precipitate was used to fill a Bruker 3.2 mm thin-wall zirconia

rotor. Silicon plugs (courtesy of Bruker Biospin) placed below the turbine cap were used to close the rotor and preserve hydration. The rotor was filled with 30 mg of wet precipitate. A

batch of freeze‐dried ANSII [U- 13C, 15N, ca. 20 mg of material] was prepared as reference sample. The protein material was packed into a Bruker 3.2 mm zirconia rotor, and rehydrated with a

solution of 9 mg/mL NaCl in MilliQ H2O, to simulate the same conditions of ANSII-AlumOH. The hydration process was monitored through 1D {1H}-13C cross-polarization SSNMR spectrum and stopped

when the resolution of the spectrum did not change any further for successive additions of the solution.26,29,30 The amount of protein present in the NMR sample of ANSII-AlumOH was

estimated to be 0.7–1 mg (per 20–25 mg of AlumOH), according to the relative intensity of 1D {1H}-13C cross-polarization spectra recorded on ANSII-AlumOH and on the sample of rehydrated

freeze-dried ANSII containing a known amount of protein. Standard 13C-detected SSNMR spectra (2D 15N 13C NCA, 2D 15N 13C NCO and 2D 13C-13C DARR, mixing time 50 ms) were acquired using the

pulse sequences reported in the literature.51 Pulses were 2.6 μs for 1H, 4 μs for 13C and 5.6 μs for 15N. The inter-scan delay was set to 1.5 s in all the experiments. The number of scans

used for the acquisition of 2D 15N 13C NCA and 15N 13C NCO experiments was 4096 and 128 for ANSII-AlumOH and rehydrated freeze-dried ANSII, respectively. Each N−C correlation experiment

collected on ANSII-AlumOH was acquired for 6 days while the 2D 13C-13C DARR (number of scans equal to 656) required 8 days of acquisition (additional experimental information is reported in

Supplementary Table 2). No significant protein desorption was observed to occur by spinning the sample at 14 kHz as proved by the comparison of the 1D {1H}-13C cross-polarization spectra

collected just after sample preparation and after the whole NMR characterization: in the two 1D {1H}-13C cross-polarization spectra the signal intensity is approximately the same

(Supplementary Fig. 1). All the spectra were processed with the Bruker TopSpin 3.2 software package and analyzed with the program CARA.52 DATA AVAILABILITY All data generated or analyzed

during this study are included in this published article and its supporting information file. REFERENCES * Glenny, A. T., Pope, C. G., Waddington, H. & Wallace, U. Immunological notes.

XVII−XXIV. _J. Pathol. Bacteriol._ 29, 31–40 (1926). Article  CAS  Google Scholar  * Del Giudice, G., Rappuoli, R. & Didierlaurent, A. M. Correlates of adjuvanticity: a review on

adjuvants in licensed vaccines. _Semin. Immunol._ 39, 14–21 (2018). Article  Google Scholar  * O’Hagan, D. T., Friedland, L. R., Hanon, E. & Didierlaurent, A. M. Towards an evidence

based approach for the development of adjuvanted vaccines. _Curr. Opin. Immunol._ 47, 93–102 (2017). Article  Google Scholar  * HogenEsch, H., O’Hagan, D. T. & Fox, C. B. Optimizing the

utilization of aluminum adjuvants in vaccines: you might just get what you want. _NPJ Vaccin._ 3, 51 (2018). Article  Google Scholar  * Sokolovska, A., Hem, S. L. & HogenEsch, H.

Activation of dendritic cells and induction of CD4(+) T cell differentiation by aluminum-containing adjuvants. _Vaccine_ 25, 4575–4585 (2007). Article  CAS  Google Scholar  * Jones, L. S. et

al. Effects of adsorption to aluminum salt adjuvants on the structure and stability of model protein antigens. _J. Biol. Chem._ 280, 13406–13414 (2005). Article  CAS  Google Scholar  *

Jully, V., Mathot, F., Moniotte, N., Préat, V. & Lemoine, D. Mechanisms of antigen adsorption onto an aluminum-hydroxide adjuvant evaluated by high-throughput screening. _J. Pharm. Sci._

105, 1829–1836 (2016). Article  CAS  Google Scholar  * Régnier, M. et al. Structural perturbation of diphtheria toxoid upon adsorption to aluminium hydroxide adjuvant. _Vaccine_ 30,

6783–6788 (2012). Article  Google Scholar  * Dong, A., Jones, L. S., Kerwin, B. A., Krishnan, S. & Carpenter, J. F. Secondary structures of proteins adsorbed onto aluminum hydroxide:

infrared spectroscopic analysis of proteins from low solution concentrations. _Anal. Biochem._ 351, 282–289 (2006). Article  CAS  Google Scholar  * Kirkitadze, M., Sinha, A., Hu, J.,

Williams, W. & Cates, G. Adjuvanted vaccine components: analysis of structure and stability. _Procedia Vaccinol._ 1, 135–139 (2009). Article  CAS  Google Scholar  * Soliakov, A., Harris,

J. R., Watkinson, A. & Lakey, J. H. The structure of Yersinia pestis Caf1 polymer in free and adjuvant bound states. _Vaccine_ 28, 5746–5754 (2010). Article  CAS  Google Scholar  *

Andreas, L. B. et al. Structure of fully protonated proteins by proton-detected magic-angle spinning NMR. _Proc. Natl. Acad. Sci. USA_ 113, 9187–9192 (2016). Article  CAS  Google Scholar  *

Quinn, C. M. et al. Dynamic regulation of HIV-1 capsid interaction with the restriction factor TRIM5α identified by magic-angle spinning NMR and molecular dynamics simulations. _Proc. Natl.

Acad. Sci. USA_ 115, 11519–11524 (2018). Article  CAS  Google Scholar  * Page, R. C., Lee, S., Moore, J. D., Opella, S. J. & Cross, T. A. Backbone structure of a small helical integral

membrane protein: a unique structural characterization. _Protein Sci._ 18, 134–146 (2009). CAS  PubMed  Google Scholar  * Soong, R. et al. Proton-evolved local-field solid-state NMR studies

of cytochrome b5 embedded in bicelles, revealing both structural and dynamical information. _J. Am. Chem. Soc._ 132, 5779–5788 (2010). Article  CAS  Google Scholar  * Park, S. H. et al.

Structure of the chemokine receptor CXCR1 in phospholipid bilayers. _Nature_ 491, 779–783 (2012). Article  CAS  Google Scholar  * Retel, J. S. et al. Structure of outer membrane protein G in

lipid bilayers. _Nat. Commun._ 8, 2073 (2017). Article  Google Scholar  * Fusco, G. et al. Direct observation of the three regions in α-synuclein that determine its membrane-bound

behaviour. _Nat. Commun._ 5, 3827 (2014). Article  CAS  Google Scholar  * Sergeyev, I. V., Itin, B., Rogawski, R., Day, L. A. & McDermott, A. E. Efficient assignment and NMR analysis of

an intact virus using sequential side-chain correlations and DNP sensitization. _Proc. Natl. Acad. Sci. USA_ 114, 5171–5176 (2017). Article  CAS  Google Scholar  * Habenstein, B. et al.

Hybrid structure of the type 1 pilus of uropathogenic _Escherichia coli_. _Angew. Chem. Int. Ed._ 54, 11691–11695 (2015). Article  CAS  Google Scholar  * Öster, C. et al. Characterization of

protein−protein interfaces in large complexes by solid-state NMR solvent paramagnetic relaxation enhancements. _J. Am. Chem. Soc._ 139, 12165–12174 (2017). Article  Google Scholar  *

Tuttle, M. D. et al. Solid-state NMR structure of a pathogenic fibril of full-length human α-synuclein. _Nat. Struct. Mol. Biol._ 23, 409–415 (2016). Article  CAS  Google Scholar  * Wiegand,

T. et al. The conformational changes coupling ATP hydrolysis and translocation in a bacterial DnaB helicase. _Nat. Commun._ 10, 31 (2019). Article  Google Scholar  * Bersch, B., Dörr, J.

M., Hessel, A., Killian, J. A. & Schanda, P. Proton-detected solid-state NMR spectroscopy of a zinc diffusion facilitator protein in native nanodiscs. _Angew. Chem. Int. Ed. Engl._ 56,

2508–2512 (2017). Article  CAS  Google Scholar  * Joedicke, L. et al. The molecular basis of subtype selectivity of human kinin G-protein-coupled receptors. _Nat. Chem. Biol._ 14, 284–290

(2018). Article  CAS  Google Scholar  * Ravera, E. et al. Solid-state NMR of PEGylated proteins. _Angew. Chem. Int. Ed. Engl._ 55, 2446–2449 (2016). Article  CAS  Google Scholar  * Fragai,

M. et al. SSNMR of biosilica-entrapped enzymes permits an easy assessment of preservation of native conformation in atomic detail. _Chem. Commun._ 50, 421–423 (2014). Article  CAS  Google

Scholar  * Ravera, E. et al. Biosilica-entrapped enzymes studied by using dynamic nuclear-polarization-enhanced high-field NMR spectroscopy. _Chemphyschem_ 16, 2751–2754 (2015). Article  CAS

  Google Scholar  * Giuntini, S. et al. Characterization of the conjugation pattern in large polysaccharide—protein conjugates by NMR spectroscopy. _Angew. Chem. Int. Ed._ 56, 14997–15001

(2017). Article  CAS  Google Scholar  * Giuntini, S., Cerofolini, L., Ravera, E., Fragai, M. & Luchinat, C. Atomic structural details of a protein grafted onto gold nanoparticles. _Sci.

Rep._ 7, 17934 (2017). Article  Google Scholar  * Jantschke, A. et al. Insight into the supramolecular architecture of intact diatom biosilica from DNP-supported solid-state NMR

spectroscopy. _Angew. Chem. Int. Ed. Engl._ 54, 15069–15073 (2015). Article  CAS  Google Scholar  * Martelli, T. et al. Atomic-level quality assessment of enzymes encapsulated in bioinspired

silica. _Chemistry_ 22, 425–432 (2015). Article  Google Scholar  * Balayssac, S. et al. Solid-state NMR of matrix metalloproteinase 12: an approach complementary to solution NMR.

_Chembiochem_ 8, 486–489 (2007). Article  CAS  Google Scholar  * Cerofolini, L. et al. Characterization of PEGylated asparaginase: new opportunities from NMR analysis of large PEGylated

therapeutics. _Chemistry_ 25, 1984–1991 (2019). Article  CAS  Google Scholar  * Wang, S. et al. Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane

protein. _Nat. Methods_ 10, 1007–1012 (2013). Article  Google Scholar  * Suiter, C. L. et al. MAS NMR of HIV-1 protein assemblies. _J. Magn. Reson._ 253, 10–22 (2015). Article  CAS  Google

Scholar  * Marchanka, A., Simon, B., Althoff-Ospelt, G. & Carlomagno, T. RNA structure determination by solid-state NMR spectroscopy. _Nat. Commun._ 6, 7024 (2015). Article  CAS  Google

Scholar  * Garçon, N. & Friede, M. 6—Evolution of adjuvants across the centuries. In _Plotkin’s Vaccines_ _(_ _Seventh Edition_ _)_ (eds Plotkin, S. A. et al.) 61−74.e4 (Elsevier,

Amsterdam, 2018). * Scarselli, M. et al. Rational design of a meningococcal antigen inducing broad protective immunity. _Sci. Transl. Med._ 3, 91ra62 (2011). Article  CAS  Google Scholar  *

Cozzi, R., Scarselli, M. & Ferlenghi, I. Structural vaccinology: a three-dimensional view for vaccine development. _Curr. Top. Med. Chem._ 13, 2629–2637 (2013). Article  Google Scholar 

* Cerofolini, L. et al. High-resolution solid-state NMR characterization of ligand binding to a protein immobilized in a silica matrix. _J. Phys. Chem. B_ 121, 8094–8101 (2017). Article  CAS

  Google Scholar  * Graham, M. L. Pegaspargase: a review of clinical studies. _Adv. Drug Deliv. Rev._ 55, 1293–1302 (2003). Article  CAS  Google Scholar  * Kumru, O. S. et al. Vaccine

instability in the cold chain: mechanisms, analysis and formulation strategies. _Biologicals_ 42, 237–259 (2014). Article  CAS  Google Scholar  * Braun, L. J. et al. Characterization of a

thermostable hepatitis B vaccine formulation. _Vaccine_ 27, 4609–4614 (2009). Article  Google Scholar  * Sastry, M., Bewley, C. A. & Kwong, P. D. Mammalian expression of isotopically

labeled proteins for NMR spectroscopy. _Adv. Exp. Med. Biol._ 992, 197–211 (2012). Article  CAS  Google Scholar  * Werner, K., Richter, C., Klein-Seetharaman, J. & Schwalbe, H. Isotope

labeling of mammalian GPCRs in HEK293 cells and characterization of the C-terminus of bovine rhodopsin by high resolution liquid NMR spectroscopy. _J. Biomol. NMR_ 40, 49–53 (2008). Article

  CAS  Google Scholar  * Vajpai, N. et al. Solution conformations and dynamics of ABL kinase-inhibitor complexes determined by NMR substantiate the different binding modes of

imatinib/nilotinib and dasatinib. _J. Biol. Chem._ 283, 18292–18302 (2008). Article  CAS  Google Scholar  * Strauss, A. et al. Efficient uniform isotope labeling of Abl kinase expressed in

Baculovirus-infected insect cells. _J. Biomol. NMR_ 31, 343–349 (2005). Article  CAS  Google Scholar  * Morgan, W. D., Kragt, A. & Feeney, J. Expression of deuterium-isotope-labelled

protein in the yeast pichia pastoris for NMR studies. _J. Biomol. NMR_ 17, 337–347 (2000). Article  CAS  Google Scholar  * Takahashi, H. & Shimada, I. Production of isotopically labeled

heterologous proteins in non-E. coli prokaryotic and eukaryotic cells. _J. Biomol. NMR_ 46, 3–10 (2010). Article  CAS  Google Scholar  * Schuetz, A. et al. Protocols for the sequential

solid-state NMR spectroscopic assignment of a uniformly labeled 25 kDa protein: HET-s(1-227). _ChemBioChem_ 11, 1543–1551 (2010). Article  CAS  Google Scholar  * Keller, R. _The Computer

Aided Resonance Assignment Tutorial (CARA)_ (CANTINA Verlag, Goldau, 2004). * Grzesiek, S. et al. The solution structure of HIV-1 Nef reveals an unexpected fold and permits delineation of

the binding surface for the SH3 domain of Hck tyrosine protein kinase. _Nat. Struct. Biol._ 3, 340–345 (1996). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This work

has been supported by Regione Toscana (CERM-TT and BioEnable), Fondazione Cassa di Risparmio di Firenze and COST Action CA15209 “European Network on NMR Relaxometry”. The authors acknowledge

the support and the use of resources of Instruct-ERIC, a landmark ESFRI project, and specifically the CERM/CIRMMP Italy center. The authors acknowledge the support of the University of

Florence and the _Recombinant Proteins JOYNLAB_. The FP7 WeNMR (project no. 261572) and H2020 West-Life (project no. 675858) European e-Infrastructure projects are acknowledged for the use

of their web portals. These portals make use of the EGI infrastructure and DIRAC4EGI service with the dedicated support of CESNET-MetaCloud, INFN-PADOVA, NCG-INGRID-PT, RAL-LCG2, TW-NCHC,

IFCA-LCG2, SURFsara and NIKHEF, and the additional support of the national GRID Initiatives of Belgium, France, Italy, Germany, the Netherlands, Poland, Portugal, Spain, UK, South Africa,

Malaysia, Taiwan and the US Open Science Grid. Funding from The Italian Ministry of Education, Universities and Research (MIUR) PRIN project no. 2012SK7ASN and the EC Contracts iNext no.

653706 is acknowledged. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Magnetic Resonance Center (CERM), University of Florence and Consorzio Interuniversitario Risonanze Magnetiche di

Metallo Proteine (CIRMMP), Via L. Sacconi 6, 50019, Sesto Fiorentino, Italy Linda Cerofolini, Enrico Ravera, Claudio Luchinat & Marco Fragai * Department of Chemistry, University of

Florence, Via della Lastruccia 3, 50019, Sesto Fiorentino, Italy Stefano Giuntini, Enrico Ravera, Claudio Luchinat & Marco Fragai * Technical R&D, GSK Vaccines, Via Fiorentina 1,

53100, Siena, Italy Francesco Berti Authors * Linda Cerofolini View author publications You can also search for this author inPubMed Google Scholar * Stefano Giuntini View author

publications You can also search for this author inPubMed Google Scholar * Enrico Ravera View author publications You can also search for this author inPubMed Google Scholar * Claudio

Luchinat View author publications You can also search for this author inPubMed Google Scholar * Francesco Berti View author publications You can also search for this author inPubMed Google

Scholar * Marco Fragai View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS M.F., F.B. and C.L. designed the research; S.G. expressed and

purified the protein and prepared the formulation; L.C., M.F., E.R., and S.G. performed the NMR experiments; L.C. performed the resonance assignment; L.C., M.F., E.R. and C.L. performed the

data analysis; all authors wrote the manuscript, read and approved the completed version. CORRESPONDING AUTHORS Correspondence to Francesco Berti or Marco Fragai. ETHICS DECLARATIONS

COMPETING INTERESTS F.B. is an employee of the GSK group of companies. All other authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE: Springer Nature remains

neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTAL MATERIAL RIGHTS AND PERMISSIONS OPEN ACCESS This article

is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you

give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material

in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative

Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a

copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Cerofolini, L., Giuntini, S., Ravera, E. _et al._

Structural characterization of a protein adsorbed on aluminum hydroxide adjuvant in vaccine formulation. _npj Vaccines_ 4, 20 (2019). https://doi.org/10.1038/s41541-019-0115-7 Download

citation * Received: 12 January 2019 * Accepted: 03 May 2019 * Published: 28 May 2019 * DOI: https://doi.org/10.1038/s41541-019-0115-7 SHARE THIS ARTICLE Anyone you share the following link

with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt

content-sharing initiative