Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection

Vaccine xxx (xxxx) xxx

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Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection Elodie Desuzinges Mandon a,1, Andrés Pizzorno b,1, Aurélien Traversier b,c, Anne Champagne a, Marie Eve Hamelin d, Bruno Lina b,e, Guy Boivin d, Emmanuel Dejean a, Manuel Rosa-Calatrava b,c,⇑,2, Anass Jawhari a,⇑,2 a

CALIXAR, 60 Avenue Rockefeller 69008, Lyon, France Virologie et Pathologie Humaine-VirPath Team, Centre International de Recherche en Infectiologie (CIRI), INSERM U1111, CNRS UMR5308, ENS Lyon, Université Claude Bernard Lyon 1, Université de Lyon, Lyon, France c VirNext, Faculté de Médecine RTH Laennec, Université Claude Bernard Lyon 1, Université de Lyon, Lyon 69008, France d Research Center in Infectious Diseases of the CHU de Quebec and Laval University, Quebec City, QC G1V 4G2, Canada e Laboratoire de Virologie, Centre National de Référence des virus Influenza Sud, Institut des Agents Infectieux, Groupement Hospitalier Nord, Hospices Civils de Lyon, Lyon F-69317, France b

a r t i c l e

i n f o

Article history: Received 14 March 2019 Received in revised form 30 September 2019 Accepted 6 October 2019 Available online xxxx Keywords: Split inactivated influenza antigen Hemagglutinin Detergent/surfactant Triton Calixarene Immunization H1N1

a b s t r a c t Influenza A viruses cause major morbidity and represent a severe global health problem. Current influenza vaccines are mainly egg-based products requiring the split of whole viruses using classical detergents such as Triton X-100, which implies certain limitations. Here, we report the use of the novel calixarene-based surfactant CALX133ACE as an alternative to classical detergents for influenza inactivated split vaccine preparation. We confirmed that CALX133ACE-based split HA antigens are fully functional and quantifiable by the ‘‘gold standard” method SRID. Additionally, as in the case of the Triton X-100-based split, the CALX133ACE-based split antigens are stable for at least 6 months at 4 °C. Moreover, immunization of mice with CALX133ACE-based split NYMC X-179A (H1N1) antigens harboring 10 to 30-fold less antigen than the commercialized trivalent inactivated vaccines VaxigripÒ or FluviralÒ induced comparable efficient protection and neutralizing antibody responses against A (H1N1)pdm09 infection. Taken together, our results demonstrate for the first time the use of a calixarene-based detergent as an efficient splitting agent for the production of optimized influenza split antigens, paving the way for significant improvement in the vaccine manufacturing process, notably with regard to the current regulation on the prohibition of endocrine disruptors, such as Triton X-100. Ó 2019 Published by Elsevier Ltd.

1. Introduction Global influenza type A and B epidemics emerge every year during winter seasons of both the northern and southern hemispheres. The WHO estimates that annual global infection rates reach 5–10% in adults and 20–30% in children, including 3–5 million cases of severe flu and up to 500,000 deaths. In addition to seasonal epi⇑ Co-corresponding authors at: CALIXAR, 60 avenue Rockefeller, 69008 Lyon, France & Virologie et Pathologie Humaine-VirPath Team, Centre International de Recherche en Infectiologie (CIRI), INSERM U1111, CNRS UMR5308, ENS Lyon, Université Claude Bernard Lyon 1, Université de Lyon, Lyon, France (M. RosaCalatrava). E-mail addresses: [email protected] (M. Rosa-Calatrava), [email protected] (A. Jawhari). 1 Co-first authors. 2 Co-last authors.

demics, (re)-emerging influenza A viruses cause recurrent pandemics. Vaccination remains the most-effective method for flu prevention and control, and neutralizing antibodies directed against surface glycoprotein antigens, mainly the hemagglutinin (HA) protein, are considered as the major effectors of protection against infection [1]. In that way, vaccination can reduce illness and severity of infection, particularly in young children and the elderly, two major risk groups [2]. Current seasonal vaccines are predominantly produced from an egg-based manufacturing process and require annual reformulation and validation to be adapted to continual drifts among circulating influenza A and B strains in both hemispheres [3]. Quadrivalent vaccine formulations contain either inactivated split or subunit antigens (FluarixÒ, GlaxoSmithKline [3]; FluzoneÒ, Sanofi Pasteur [4]), or live attenuated viruses (FlumistÒ, MedImmune [5]) derived from two influenza A strains and two influenza B strains. In order to complement egg-based

https://doi.org/10.1016/j.vaccine.2019.10.018 0264-410X/Ó 2019 Published by Elsevier Ltd.

Please cite this article as: E. D. Mandon, A. Pizzorno, A. Traversier et al., Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.018

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processes, increase production capacity of manufacturers and accelerate the delivery of potential pandemic influenza vaccines, alternative production systems were developed (FluBlokÒ, Protein Sciences/Sanofi [6]; FlucelvaxÒ, Novartis [7]) [8,9]. Split inactivated vaccines contain influenza viral particles that are disrupted and solubilized by detergent treatments. The ability of detergents to split and solubilize virus particles with preserved and stabilized antigen structures allow split-based vaccines to induce efficient immunogenicity, while displaying lower reactogenicity in comparison to inactivated whole viruses [10]. Split influenza vaccines contain between 9 and 15 lg of each monovalent HA protein [11], respectively, whether they are administered intradermally or intramuscularly [12,13]. A more immunogenic formulation (Fluzone High doseÒ) containing a higher dose of HA (60 lg) is also available for specific population groups, specially the elderly. Nevertheless, although this egg-based split antigen strategy accounts for the vast majority of the current worldwide influenza vaccine production capacity (1–1.5 billion doses), it presents several limitations and drawbacks. The dependence on a massive embryonated egg supply, long timeline of vaccine manufacturing and delivery, or the reduced production yield of certain influenza strains, are a few examples that underscore the need to maximize the optimization of the egg-based process. In that regard, improving the immunogenicity and efficacy of split antigens remains a major challenge. The physical-chemical proprieties of detergents (headgroup and tail), which are variable from one detergent to another, are critical for solubilization and stabilization efficiency, therefore conditioning the native structural integrity and conformation of proteins of interest. Indeed, structural stability of proteins is of crucial importance for an efficient presentation of antigenic peptides on the major histocompatibility complex (MHC), playing a decisive role on the triggering of effective immune responses [14]. Nonionic detergent Triton X-100 and ionic detergent sodium deoxycholate have been typically used since 1970 s in the production of inactivated influenza vaccines in order to disrupt and solubilize the lipid virus envelope containing the HA, Neuraminidase (NA) and Matrix 2 (M2) viral antigens. Besides such ‘‘classic” detergents, new families of amphiphilic reagents have recently emerged [15]. For instance, novel calixarene-based detergents/surfactants with critical micelle concentrations (CMCs) ranging from 0.05 to 1.5 mM have been successfully used to solubilize, purify and stabilize different functional membrane proteins [15–19]. These novel detergents were initially designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins, in addition to p-stacking interactions between the calixarene platform and aromatic residues [20]. Such properties lead to a better preservation of the structural and functional integrities of membrane proteins. In a previous proofof-concept study with influenza viral protein, we have described the use of a calixarene detergent CALX3 for the solubilization and purification of a native and functional influenza A tetrameric M2 proton selective ion channel from H1N1 infected MDCK cell membranes (17). Here, we tested another calixarene-based surfactant, named CALX133ACE, with the objective of exploring a novel detergent-based antigen splitting method, as a putative alternative to current detergents like Triton X-100 or sodium deoxycholate for the preparation of influenza inactivated split vaccines, notably with regard to the current REACH Regulation 1907/2006/EC on the potential prohibition of endocrine disruptors [21]. Our results demonstrate that CALX133ACE efficiently splits whole influenza viruses and preserves the antigenic properties of the HA, as measured by the ‘‘gold standard” Single Radial Immuno-Diffusion (SRID) quantitative assay. Moreover, the CALX133ACE-based monovalent H1N1 influenza split confers a strong anti-HA neutral-

izing antibody response and protects mice against lethal challenge with influenza A(H1N1)pdm09 virus. Most important, immunization with the CALX133ACE-based split harboring an HA antigen dose tenfold lower than that of the commercialized trivalent inactivated vaccines VaxigripÒ or FluviralÒ retains complete protection effectiveness and induces comparable neutralizing antibody responses. Additionally, the stability of this new vaccine preparation at 4 °C has been validated for at least 6 months. Taken together, the results of our proof-of-concept study pave the way for further investigations aiming to fully assess the potential of calixarene-based antigen splitting methods to improve both vaccine response efficacy and manufacturing processes.

2. Experimental procedures Virus production in egg, purification and concentration – H1N1 A/California/7/2009 virus (A/H1N1 pdm09) and H1N1 X179A vaccine strain (NYMC X-179A; NIBSC code: 11/112) were grown in 11 day-old embryonated hen eggs (Specific PathogenFree Leghorn strain) at 34 °C for 72 h. Allantoic fluid was harvested and clarified by centrifugation at 2000 rpm for 20 min at 4 °C. The supernatant was collected and pelleted by ultra-centrifugation at 164,138g for 2 h at 4 °C. Virus pellets were resuspended in pH = 7.40 phosphate buffered saline (PBS) and loaded onto a 25– 60% sucrose gradient. The gradient was centrifuged at 164,138g for 12 h. The virus fractions were harvested and further pelleted at 164,138g for 3 h. Virus splitting and inactivation – Concentrated and purified NYMC X-179A virus was splitted in 2.5 M KCl phosphate buffer containing either 0.5% or 1% Triton X-100 (Sigma-Aldrich) for 1 h at 37 °C or 0.5% or 1% CALX133ACE for 2 h at 4 °C. The split products were then dialyzed with PBS with stirring overnight at 4 °C to eliminate the KCl and CALX133ACE. Triton was removed with Bio-Beads (Bio-Rad) after stirring during 40 min at room temperature. Split products were inactivated with a final concentration of 0.01% formaldehyde during 72 h at 20 °C. Final split products were stored at 4 °C. Hemagglutination Assay (HA) – HA titers in split preparations were measured by standard hemagglutination assay in v-bottom 96-well plates as described previously [22–24]. Briefly, 50 ml aliquot samples of vaccine preparations were serially diluted 1:2 in PBS and incubated with 50 ml of 0.5% chicken red blood cells (RBCs) for 1 h at room temperature. Mean HA titers corresponding to the reciprocal of the last dilution where hemagglutination was observed (absence of RBC precipitation) were expressed in HA units/50 ml. HA titers were also measured as a surrogate of the stability of the H1N1 X-179A virus split. Single Radial Immunodiffusion Assay (SRID) – The amount of specific antigen (H1N1) as a correlate of vaccine potency was measured in vitro by single-radial-immunodiffusion assay (SRID) as the ‘‘gold standard” method for vaccine manufacturing. An indubiose A37 1% gel (PALL France) containing anti-HA polyclonal serum (15 ml in a 1 ml gel) was poured on a slab of glass and wells were pierced. Standard antigen (09/196) and polyclonal serum (12/108) were obtained from the National Institute for Biological Standards and Control (NIBSC). The reference HA standard (37 mg/ml) and samples were incubated with a final concentration of 1% of Zwittergent 3-14 (MERCK) for 30 min at room temperature, then diluted at 1:1; 3:4; 1:2; 1:4 and loaded in triplicate into wells. Gels were incubated for 20 h at 22 °C in a wet room to enable antigen migration. Gels were then washed in saline solution dried firstly under absorbent paper for 30 min and then at 60 °C for 20 min. Slabs were finally stained for 8 min in Coomassie brillant blue R (COGER). Precipitin rings were measured using ProtoCol 3 (SYNBIOSIS) and compared to the standard. Potency was expressed

Please cite this article as: E. D. Mandon, A. Pizzorno, A. Traversier et al., Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.018

E.D. Mandon et al. / Vaccine xxx (xxxx) xxx

in mg HA/ml. Viral production, splitting procedure, SRID and HA assays were conducted in duplicate according to established protocols [24]. CALX133ACE quantification by mass spectrometry – Mass spectrometry was performed using a Voyager-DE Pro MALDI-TOF Mass Spectrometer (AB Sciex, Framingham, MA) equipped with a nitrogen UV laser (k = 337 nm, 3 ns pulse) as previously described [25]. The instrument was operated in the positive reflectron mode (mass accuracy: 0.008%) with an accelerating potential of 5 kV. All volumes were weighted on a precision scale to maximize accuracy and compensate for pipetting errors. Typically, 1 ll of detergentcontaining sample was added of 9 ll of 10 g CHCA/L 50:50 acetonitrile:water. 1 ll of 10 g NaI/L acetone was added to the detergent/matrix mixture to produce MNa + cations. One microliter of the final solution was laid on the MALDI target and air-dried before analysis. For each trial, mass spectra were obtained by accumulation of three series of 300 laser shots, each acquired in 3 distinct areas of the dried mixture. Antigen quantification by mass spectrometry. Quantification of Hemagglutinin (HA) antigen was performed by mass spectrometry LC-MS/MS based on previously described work [26]. Typically, vaccine samples were diluted using 50 mM bicarbonate buffer pH8.0 complemented with labelled peptides. Samples were treated with DTT (20 mM final) and heated for 20 min at 60 °C. After a cooling step, Iodoacetamide (50 mM final) was added for 40 min in the dark followed by trypsin digestion overnight. Samples were finally analyzed by LC-MS/MS. Tunable Resistive Pulse Sensing (TRPS) analysis – Quantification and charge/size analyses of selected Triton X-100- and CALX133ACE-based formulations were performed using the qNano Gold platform (Izon Science, Oxford, UK) combining tunable nanopores with proprietary data capture [27]. Briefly, Triton X-100- and CALX133ACE-based formulations were prepared as previously indicated, and then diluted in PBS. Samples were measured using the nanopore NP200 (Izon Science, Oxford, UK) at a 45 mm stretch (0.78 V and 10 mbar). The concentration of particles was standardized using multi-pressure calibration with 70 nm carboxylated polystyrene beads provided by Izon Science. Particles were detected as short current pulses and data analysis was carried out using the Izon Control Suite software v3.3 (Izon Science, Oxford, UK). SDS-PAGE and Western-Blot – Proteins were denatured by 2% SDS and separated by SDS-PAGE on a 4–15% acrylamide gel (4– 15% Mini-PROTEANÒ TGX Stain-FreeTM Gel, Bio-Rad, cat#4568085). The acrylamide gel was then activated by UV light with a ChemiDocTM MP system (Bio-Rad, cat#170-8280) for Stain-FreeTM detection, or stained using Oriol staining (BioRad, cat#161-0496). For western-blotting, proteins were subsequently immobilized by electro-transfer to PVDF membrane using the trans-blot turbo transfer system (Bio-Rad, cat#170-4155). The immunodetection of HA, NA and M2 proteins was performed by using the SNAP i.d. system (Millipore, cat#WBAVDBASE) with an anti-HA antibody (3:500; SantaCruz Biotechnology, cat# sc-52025), an anti-NA antibody (3:200; SinoBiologicals, cat#11058-V07B) or an anti-M2 antibody (3:200; SantaCruz Biotechnology, cat# Sc32238). Cross-link experiment – 2 ml of samples were incubated with 8 ml of glutaraldehyde solution to reach a final concentration of 0%, 0.01%, 0.02% and 0.05%. Each sample was then analyzed by SDS-PAGE and western-blot as described above. Clear-native-PAGE and Western-Blot – Protein samples were separated on a 4–15% acrylamide gel (4–15% Mini-PROTEANÒ TGX Stain-FreeTM Gel, Bio-Rad, cat#456-8085) by migration in a Tris-Glycine buffer (Euromedex, cat#EU0550). The acrylamide gel was then activated by UV light with a ChemiDocTM MP system

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(Bio-Rad, cat#170-8280) for Stain-FreeTM detection. Proteins were subsequently immobilized by electro-transfer to PVDF membrane using the trans-blot turbo transfer system (Bio-Rad, cat#1704155). The immunodetection of HA protein was performed by using the SNAP i.d. system (Millipore, cat#WBAVDBASE) with an anti-HA antibody (3:500; SantaCruz Biotechnology, cat# sc52025). Transmission Electron Microscopy (TEM)– Suspensions were adsorbed on 200 Mesh copper grids coated with formvar-C for 2 min at RT. Then grids with suspension were colored with 3% uranyl acetate for 1 min and observed on a transmission electron microscope (Jeol 1400 JEM, Tokyo, Japan) equiped with a Gatan camera (Orius 600) and Digital Micrograph Software. In Vivo studies – Animal protocols were approved by the Institutional Animal Care Committee of the Centre Hospitalier Universitaire de Québec (CPAC protocol authorization #2013-134) according to the guidelines of the Canadian Council on Animal Care. All animal studies were carried out in 6–8-week-old female BALB/c mice (Charles River Laboratories, QC, Canada). Mice received a clinical inspection on arrival and were housed in micro-isolator cages (4 mice per cage) under standard biosafety 2 conditions: room temperature (22 ± 2 °C), hygrometry (55 ± 10%), light/dark cycle (12 h/12 h, light 7.15 a.m. to 7.15p.m.), air replacement: 15–20 volumes/h, food (T2018.sx, Harlan Teklad) and drinking water ad libitum. All animals were allowed to acclimate to environmental conditions for 7 days prior to the beginning of the experiments and then randomized in groups of 6, 8 or 12, as indicated on each case. Animals were immunized with either experimental or control vaccine formulations by two three-week-apart (Day -42 and Day -21) 0.1 ml intramuscular (i.m.) injections in the same hind limb. For the commercial trivalent inactivated vaccines (TIV) used as positive controls of the experimental challenge setup, starting from an initial quantity of 15 mg per strain as stated in the label and also confirmed by in-house SRID and mass spectrometry, dilutions containing a final HA amount of 3 mg per strain were prepared. On Day 0, three weeks after the second (boost) immunization, mice were lightly anesthetized with isoflurane, and then infected by intranasal (i.n.) instillation of an influenza A/California/7/2009 (H1N1) virus in 30 ml of PBS. A preliminary experiment determined the lethal dose 50% (LD50) of the chosen virus to be 103 PFU/mouse, and a challenge dose of 2LD50 was selected in order to evaluate the potential protective effect of vaccine candidates. After the challenge, animals were followed at least once a day for mortality, body weight and clinical signs such as lethargy, ruffled fur and/or hunched posture until death or sacrifice at Day 14. Animals were euthanized if they reached the humane endpoint of >20% weight loss. In addition, blood samples were collected from the submandibular vein of each animal on Day 42 (before the first immunization), 21 (before the second immunization), and 0 (before challenge). After clotting and centrifugation, sera were divided into 2 aliquots and stored at 20 °C for subsequent Hemagglutination Inhibition (HAI) and Microneutralization (MN) assays. Both serologic tests were performed against the same virus used for the viral challenge (A/California/7/2009 (H1N1)), following the standard WHO guidelines [24]. Pulmonary viral titers–Depending on the total number of mice per group used in each protocol (6, 8 or 12), 2, 3 or 4 randomly selected mice per group were sacrificed under isoflurane anesthesia at the indicated time points for the determination of lung viral titers. Lungs were removed aseptically and then homogenized in Dulbecco’s modified Eagle medium (Life Technologies Corporation, Grand Island, NY) with antibiotics, using a potter homogenizer (Heidolph, RZR 2020). Cells were pelleted by centrifugation (2000g for 5 min), and supernatants were titrated by plaque assays

Please cite this article as: E. D. Mandon, A. Pizzorno, A. Traversier et al., Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.018

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in ST6GalI-MDCK cells. The investigator was blinded to animal group allocation. 3. Results 3.1. CALX133ACE as a novel detergent for influenza antigen vaccine preparation The main goal of this study was to evaluate the potential of novel calixarene based detergents as alternative splitting agents for egg-based influenza virus vaccine strains, in comparison with a classic detergent, such as Triton X-100. Two calixarene candidates, CALX133ACE and CALX1103ACE, sharing the same calixarene platform (with carboxylate groups on the top and a hydrophobic tail) but differing in the length of their hydrophobic tail (3 or 10 carbons, respectively) were initially tested (Fig. 1A), following the study design summarized in Fig. 1B. Briefly, we performed a standard egg-based production of the NYMC X-179A (H1N1) vaccine seed. Each antigen preparation was generated from the same initial quantity of concentrated and purified virus, including either dialysis or Bio-beads-based detergent removal and viral inactivation steps, as described in Materials and Methods. Triton X100 (1%) and calixarene (1%)-based antigen preparations were first analyzed by native and SDS PAGE western blot, and then by standard hemagglutination [28] and SRID [22] assays. Western blot analysis showed that either CALX133ACE, CALX1103ACE, or Triton X-100 detergents (Fig. 1C) successfully solubilized the three forms of hemagglutinin (HA0, HA1 and HA2) at comparable rates. Indeed, a stronger signal was observed in the solubilized fraction (S) in comparison to the pellet (P) (Fig. 1C, compare lane 2 to 1, 4–3 and 6–5). Comparable solubilization efficiencies were also obtained for the neuraminidase (NA) and the M2 ion channel (Fig. 1C). We additionally performed native PAGE to ensure that solubilized fractions contained oligomeric viral antigens, and observed trimeric HA, trimeric NA, and tetrameric M2 proteins for all solubilization conditions (Fig. 1D, compare lanes 1–6 for HA, 7–12 for NA and 13–18 for M2, and Fig. S3). Most importantly, high HA titers were measured from both Triton X-100 and CALX133ACE-based H1N1 antigen preparations (32,768 and 16,384 UHA/50 ml, respectively, Fig. 1E), hence validating HA functionality. In contrast, no HA titer could be determined for the CALX1103ACE-based preparation, owing to the CALX1103ACE-induced lysis of erythrocytes (data not shown). Considering this observation and the fact that, contrary to CALX133ACE, CALX1103ACE interfered with serial immunodiffusion assay (data not shown) and was not efficiently dialyzable as well, we decided that CALX1103ACE detergent was not suitable for a split vaccine antigen preparation and therefore we only pursued the evaluation of the CALX133ACE. In that regard, Fig. 1E and F show that CALX133ACE enabled successful HA quantification with the ‘‘gold standard” SRID assay (68.37 lgHA/ml) without interfering with antigen immunodiffusion. Similar results were also obtained for CALX133ACE-based H3N2 and B antigen preparations (Table S1). Overall, our results suggested that HA was fully functional and immunogenic in CALX133ACE-based antigen preparation, as well as within Triton X-100-based preparation. 4. Biochemical and biophysical characterization of the CALX133ACE-based influenza antigen preparation To better characterize the CALX133ACE-based HA antigen preparation, we first used Tunable Resistive Pulse Sensing (TRPS). TRPS is an impedance-based system. Typically, a voltage is applied across a pore that is filled with electrolyte, resulting in an ionic current. As particles cross the pore they briefly increase electrical

resistance, creating a resistive pulse, which is precisely proportional to particle volume. As shown in Fig. 2A and B, objects resulting from virus antigen solubilization with either Triton X-100 or CALX133ACE exhibit distinct profiles. The nanoparticle rate from the CALX133ACE preparation (1022.1p/min) is up to 10 times faster to those from the Triton X-100 preparation (121.4 p/min), certainly due to the ionic charge of CALX133ACE. Additionally, the CALX133ACE preparation harbors a more heterogeneous size distribution of particle species. Indeed, we observed a bimodal distribution with a very high proportion for 70- and 110 nm-sized particles, and a mean diameter of 105 nm (Fig. 2A). In contrast, the Triton-X100 preparation shows an apparent higher homogeneity, containing mainly 70 nm-sized particles and a mean diameter of 83 nm (Fig. 2B). To gain further insight into the behavior of the HA antigens within the CALX133ACE-based preparation, chemical crosslinking and SDS-PAGE/western-blot analysis were performed. Much higher molecular weight species were observed within the CALX133ACE preparation when high concentration of chemical crosslinker (glutaraldehyde) was used (0.01, 0.02 and 0.05%). Conversely, trimeric HAs were mainly observed in the Triton X-100 split preparation (Fig. 2C, compare lanes 3–4 to 7–8, respectively). These results were confirmed by Native PAGE coupled to anti-HA immunoblot analysis. As observed in Fig. 2D, the predominantly high molecular weight species within the CALX133ACE-based preparation are not able to enter the native gel, whereas HA trimeric and monomeric forms are mainly detected from the Triton X-100 preparation (compare lane 1–2, Fig. 2D). In line with these results, negative stain electron microscopy images of the CALX133ACE-based preparation (Fig. 2E and F) clearly highlighted disrupted and heterogeneous membrane envelope structures. Such patterns are usually observed within inactivated split influenza virus preparations that contain embedded HA trimers, protein aggregates and elongated ribonucleoprotein particles [23,29]. Interestingly, whereas very large disrupted virus particles of up to 170 nm were observed (Fig. 2E), few or no HA rosettes could be identified. Overall, CALX133ACE and Triton X-100 splitting resulted into different profiles. This was also confirmed by Dynamic Light Scattering (DLS) (Fig. S1). Indeed, two main populations of a diameter of 159 and 290 nm were detected for CALX133ACE-based split. While for Triton X-100 based preparation, much smaller species of 11–15 nm were observed. Altogether, our results suggest that CALX133ACE surfactant leads to the production of larger and more heterogeneous membrane-embedded HA particles. Importantly, this observation would not correspond to antigen aggregates, since there is no indication of protein misfolding, but rather to fully functional HA antigens, as illustrated by hemagglutination and SRID assays (Fig. 1E and F). Altogether, these results advocate for the property of CALX133ACE surfactant to be a splitting agent, as efficient as the standard Triton X-100, to produce inactivated influenza vaccines.

5. CALX133ACE-based split inactivated influenza antigens confer a strong anti-HA neutralizing antibody response and protect mice against lethal A/H1N1 pdm09 infection challenge Following initial characterization, the next step was to evaluate the capacity of the CALX133ACE-based monovalent (H1N1) influenza split to induce a significant anti-HA neutralizing antibody response and to efficiently protect mice against a lethal influenza A(H1N1)pdm09 virus challenge. As indicated in Fig. 3, BALB/c mice (n = 12/group) were immunized twice (Day-42 and Day-21) with either a Triton X-100-based split containing 3 mg of HA or CALX133ACE-based splits containing 0.5 or 3 mg of HA. A positive immunization control group, immunized with an equivalent dose of 3 mg of HA of the commercial TIV FluviralÒ was included to

Please cite this article as: E. D. Mandon, A. Pizzorno, A. Traversier et al., Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.018

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A

O

B

CO2Na CO2Na

NaO2C

Virus production Egg based A/H1N1 purified and concentrated

OH OH HO

Virus splitting (Triton X-100 or CALX-R compound)

R

Triton X-100

C

CALX

SDS-PAGE

Detergent removal/ Cencentration / Inactivation

Triton X-100 CALX133ACE CALX1103ACE

MW P S

P S

P S

95 72 55 44 34 26

HA0 HA1

Functional characterisation (Standard hemagglutination and single radial immunodiffusion assays)

HA2

72 55

NA

17

M2 1

2

D

3

4

5

In vivo evaluation (mice) (Neutralizing antibody response and protection assays)

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Triton X-100 CALX133ACE CALX1103ACE

Triton X-100 CALX133ACE CALX1103ACE

MW P S 250 170

P S

MW P S

P S HA trimer

95 43

2

3

4

5

Triton X-100 CALX133ACE CALX1103ACE

P S

P S

NA trimer

170

HA monomer

1

Native-PAGE

Native-PAGE

Native-PAGE

*

130 95 43

6

7

8

9

10

11

MW P S 170

P S

P S

130 95 72 55 43

12

M2 tetramer

13

14

15

16

17

18

F

E

1/1 Purified virus

Triton X-100 4% CALX133ACE 1%

8192

32768 16384

(µg HA/ ml)

(µg HA/ ml)

Residual detergent

108.88 68.37

108.88 82.30

0.15 0.03

3/4

1/2

1/4

Triton X-100 CALX 133ACE

Fig. 1. Splits of egg-based influenza vaccine antigen using Triton X-100 and Calixarene-based detergents. (A) Chemical structure of Triton X-100 and calixarenes based detergent (CALX). R- represents an alkyl chain of variable length (CALX133ACE and CALX1103ACE alkyl chains contain 3 and 10 carbons, respectively). (B) Work plan used to generate and analyze fragmentation of purified egg-based NYMC X-179A (A(H1N1)) virus using triton-X100 or CALX-R compound. This includes virus production, virus fragmentation, dialysis/ultrafiltration/formaldehyde inactivation, in vitro hemagglutination (HA) and single radial immunodiffusion assays (SRID), and in vivo validation (HA inhibition and microneutralization assays, and measure of mice survival rate). (C) SDS-PAGE showing influenza vaccine solubilization efficiency by using CALX compounds and Triton-X100. 10 ml of each pellet and supernatant fractions were loaded at equivalent volume. (D) Native PAGE showing influenza vaccine solubilization efficiency of HA trimer, NA trimer and M2 tetramer by using CALX compounds and Triton-X100. * corresponds to possible maturation or degradation product. (E) Quantification of HA antigen by hemagglutination (UHA/50 ll) and single radial immunodiffusion assays (SRID, lgHA/ml), from Triton X-100- and calixarene-based antigen preparations. (F) Observation of antigen precipitation rings in the SRID assays. Antigen preparations were diluted as indicated and loaded onto agarose gel containing polyclonal antiserum obtained from NIBSC and generated against egg-derived A/California/07/2009 HA. P and S correspond to pellet and supernatant, respectively.

validate the protective capacity of strain-matched vaccination in the conditions of the experimental challenge setup. Two Tritonbased (‘‘Mock Triton X-100”) or CALX133ACE-based (‘‘Mock CALX133ACE”) negative controls lacking viral antigens were also included. Three weeks after the second immunization (Day 0), mice were infected with two LD50 of influenza A/California/7/2009 (H1N1)pdm09 virus, and mortality, weight loss and clinical signs were recorded daily for 14 days (Fig. 3A). As expected, two immunizations with FluviralÒ or Triton X-100 based monovalent split H1N1 trigger a significant anti-HA specific neutralizing antibody response at Day 0, as observed in Hemagglutination Inhibition (HAI) and Microneutralization assays (MN) using A/California/7/2009 (H1N1)pdm09 virus (Fig. 3B and C). Similarly, both CALX133ACE-based splits, containing either 0.5 or 3 mg of HA, induce high HAI and MN titers at Day 0. Interestingly, HAI and

MN titers obtained with 3 mg of HA from CALX133ACE-based split are significantly higher than those obtained with the Triton X-100based monovalent split and FluviralÒ (HAI titers only), containing an equivalent quantity (3 mg) of H1 hemagglutinin (Fig. 3B and C). Moreover, similar results were obtained after immunization with the low HA CALX133ACE-based split (containing 0.5 instead of 3 mg) when compared to those induced by the 3 mg dose of the FluviralÒ or Triton X-100 based monovalent splits H1N1. Noteworthy, ‘‘Mock Triton X-100” and ‘‘Mock CALX133ACE” negative control formulations failed to induce an anti-HA neutralizing response (Fig. 3B and C). As expected, infection of mockimmunized groups resulted in progressive weight loss, with all mice reaching the 20% humane endpoint (0% survival) by Day 7 post-challenge (Fig. 3D and E). Conversely, immunization with either FluviralÒ or Triton X-100 based monovalent splits resulted

Please cite this article as: E. D. Mandon, A. Pizzorno, A. Traversier et al., Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.018

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Fig. 2. Biochemical and biophysical characterization of influenza antigen preparations. (A and B) Size distribution and concentration (particles/ml) of antigen nanoparticles from Triton X-100 (A) and CALX133ACE (B) preparations measured by Tunable Resistive Pulse Sensing (TRPS). Means of measured particle diameters, particle rate (/min) and number (n) are also indicated. (C) Chemical crosslinking of CALX133ACE and Triton X-100 antigen preparations using increasing concentrations of glutaraldehyde (0, 0.01, 0.02 and 0.05%). Proteins were separated on SDS-PAGE and immunodetected with anti-HA antibody. High molecular weight species, not able to enter the gel, are indicated (asterisks). (D) Native-PAGE analysis of the antigen preparations by using anti-HA antibody immunodetection. High molecular weight species (asterisks) are mainly observed with the CALX133ACE-based preparation. Representative electron micrographs of negatively stained CALX133ACE-based (E) and Triton X-100 (F) antigen preparations. The micrographs show particles that correspond to splitted-virions clearly harboring embedded HA trimers (black for CALX133ACE and red or blue arrows, for Triton X-100 based preparations). HA trimers were also immuno-stained and nicely distributed as solubilized protein (outside of the viral membrane) only in Triton X-100 based condition. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

in 100% survival and no significant weight loss was observed after viral challenge with a lethal dose. Moreover, the two CALX133ACEbased splits containing either 0.5 or 3 mg of HA also conferred full protection (100% survival) and no significant weight loss (Fig. 3D and E). In line with these results, whereas mean infectious lung viral titers at Day 4 post-challenge were significantly lower in the Triton X-100 based monovalent split group compared to the Mock vaccinated groups, no infectious virus was detected in the lungs of mice immunized with either FluviralÒ or the CALX133ACE-based (0.5 or 3 mg of HA) splits (Fig. 3F). Altogether, these results confirm the complete protective capacity of the CALX133ACE-based influenza virus split preparation, including that with lower dose of HA. 6. CALX133ACE-based split inactivated influenza preparation retain effectiveness at a tenfold lower HA antigen dose. To further challenge the effectiveness of the CALX133ACE-based split inactivated influenza antigens, we prepared new split formu-

lations containing different combinations of antigen quantity (0.5, 0.3 or 0.1 mg of HA) and CALX133ACE detergent (1% and 0.5%). The first detergent concentration (1%) was selected in order to be in line with that of Triton X-100 and of our previous experiment (Fig. 3), whereas the lower concentration (0.5%) was selected bearing in mind the long-term objective of minimizing the residual amount of detergent in the final antigen preparation of a commercial vaccine. We then performed a dose-response protection study by immunizing mice, as shown in Fig. 4. All split preparations were successfully quantified by hemagglutination and SRID assays, which confirmed the full immunogenic properties of the 0.5% CALX133ACE-based splits (Fig. 4A). Following an experimental approach analogous to the one described in Fig. 3, groups of 8 mice were immunized twice (Day-42 and Day-21) with either 1% and 0.5% Triton X-100-based splits containing 0.5 mg of HA, or 1% and 0.5% CALX133ACE-based splits containing 0.5, 0.3 or 0.1 mg of HA (Fig. 4B). Groups of mice immunized with 3 mg of HA from commercial TIV FluviralÒ or with PBS as positive or negative (mockimmunized) controls, respectively. An additional positive control

Please cite this article as: E. D. Mandon, A. Pizzorno, A. Traversier et al., Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.018

E.D. Mandon et al. / Vaccine xxx (xxxx) xxx

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Fig. 3. CALX133ACE-based influenza vaccine antigen split confers strong antibody response and protects mice against lethal viral challenge. (A) Schematic representation of the immunization/viral challenge experimental protocol. 6–8-week-old female BALB/c mice (n = 12/group) were vaccinated with Triton-X-100n-based (3 mg of HA) or CALX133ACE-based (3 or 0.5 mg of HA) influenza split preparations and then infected with influenza A/California/7/2009 (H1N1) virus. Fluviral TIV (GSK, 3 mg of HA) and Triton-based (n = 6/group) or CALX133ACE-based (n = 6/group) preparations without viral antigens were used as positive and negative vaccine controls, respectively. (B and C) Hemaglutination inhibition (HAI) and microneutralization (MN) titers (±SEM) against influenza A/California/7/2009 (H1N1). Titers are expressed as the reciprocal of the limit dilution (n = 3/group). (D and E) Percent survival and mean (±SEM) weight loss during the 14-day post-challenge follow up period. (F) Median lung viral titers (n = 4/group or 2/group for case and control groups, respectively) on day 4p.i. **P < 0.01 and ***P < 0.001 for differences compared by Log-rank (Mantel-Cox) Test (survival) or one-way ANOVA with Tukey’s post-test (weight loss).

group immunized with the Triton X-100-based commercial TIV VaxigripÒ (3 mg of HA) was also included. Once again, two immunizations were necessary to induce a detectable influenza-specific antibody response, with comparable HAI and MN titers between the two positive controls (Fluviral and Vaxigrip TIV) and all the Triton X-100- or CALX133ACE-based preparations. As expected, no serologic response was detected in PBS-vaccinated animals (Fig. 4C and D). All tested preparations conferred complete protection (100% survival) against the viral challenge, compared to the 20% survival observed in the PBS-vaccinated group. Moreover, while maximum mean relative weight losses of 5% were observed among all vaccinated groups, they reached the 20% humane endpoint in the PBS-vaccinated group (Fig. 4E and F). Pulmonary viral titers (Fig. 4G) showed that both Triton X-100-based (0.5 and 1%) preparations as well as the 0.5% CALX133ACE-based formulation

containing 0.1 mg of HA failed to significantly reduce viral load on Day 4 post-challenge. Conversely, 0.5 or 0.3 mg of HA antigen in CALX133ACE-based formulations were sufficient to induce a significant reduction of viral load comparable to that observed in the FluviralÒ and VaxigripÒ groups (immunized with 3 mg of HA). Taken together, our results demonstrate the relevance of CALX133ACEbased influenza vaccine preparation compared to currently used TIV vaccines, even at a tenfold lower HA antigen dose.

7. Discussion The intrinsic recurrent genetic shifts and drifts of the influenza virus represent a constant challenge for the implementation of effective preventive and therapeutic measures, clearly exemplified

Please cite this article as: E. D. Mandon, A. Pizzorno, A. Traversier et al., Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.018

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Fig. 4. CALX133ACE-based influenza vaccine candidate retains complete effectiveness at a tenfold lower HA antigen dose. (A) Characterization of the Triton-based and CALX133ACE-based influenza split antigens used for mice immunization. (B) Schematic representation of the immunization/viral challenge experimental protocol. 6–8-weekold female BALB/c mice (n = 8/group) were vaccinated with Triton-X-100-based or CALX133ACE-based influenza split preparations containing different combinations of detergent percentage (1 or 0.5%) and antigen quantity (0.5 mg of HA for Triton-X-100 and 0.5, 0.3 or 0.1 for CALX133ACE) and then infected with influenza A/California/7/2009 (H1N1) virus. Fluviral TIV (GSK, 3 mg of HA), Vaxigrip TIV (Sanofi Pasteur, 3 mg of HA) and PBS were used as positive and negative vaccine controls, respectively. (C and D) Hemaglutination inhibition (HAI) and microneutralization (MN) titers (±SEM) against influenza A/California/7/2009 (H1N1). Titers are expressed as the reciprocal of the limit dilution (n = 3/group). (E and F) Percent survival and mean (±SEM) weight loss during the 14-day post-challenge follow up period. (G) Median lung viral titers (n = 3/group) on day 4 p.i. **P < 0.01 and ***P < 0.001 for differences compared by Log-rank (Mantel-Cox) Test (survival) or one-way ANOVA with Tukey’s post-test (weight loss).

by the need of annually manufacturing very high quantities of vaccine doses containing adapted influenza strains [30]. Currently, the egg-based split antigen strategy is the most commonly used for worldwide influenza vaccine manufacturing. Although efficient, this process presents several limitations and drawbacks that require optimization in order to improve the immunogenicity and efficacy of the vaccine inactivated split-antigens [31]. Moreover, latest amendment of the ‘Authorization List’ included in the REACH Regulation 1907/2006/EC that concerns all industrial manufacturers in life sciences [21], directly affects the widely used viral inactivation split agent Triton X-100. Indeed, as part of the family of octylphenol ethoxylate compounds listed for their endocrine disrupting properties for the environment, the use of Triton X100 shall be prohibited by 2021, hence prompting vaccine manufacturers to develop alternatives. The main objective of our study was to evaluate a novel calixarene-based detergent CALX133ACE as an alternative to the classic detergent for influenza inactivated split vaccine preparation. We confirmed for the first time the capacity of CALX133ACE to efficiently split whole H1N1, H3N2 and B influenza viruses,

while completely preserving the antigenic properties of the HA, as measured by the ‘‘gold standard” quantitative method SRID (Fig. 1E and F and Table S1). Additionally, we showed that CALX133ACE-based split inactivated influenza preparation contains only residual amounts of CALX133ACE surfactant (0.01%), as determined by mass spectrometry. Most important, we report that the immunization of mice with a CALX133ACE-based monovalent H1N1 influenza split preparation, harboring 10 to 30-fold less antigen than the commercialized trivalent inactivated vaccines VaxigripÒ or FluviralÒ, confers a strong and comparable anti-HA neutralizing antibody response as well as complete protection against a lethal challenge with influenza A(H1N1)pdm09 virus. Several hypotheses could explain such promising properties of the CALX133ACE molecule. The nature of the CALX133ACE per se could confer putative inherent immunostimulatory properties. However, our results indicating that addition of increasing quantities of CALX133ACE into standard TIV preparations did not result in increased HAI or MN titers after mice immunization (data not shown), suggest an absence of such potential immunomodulatory effect of CALX133ACE, at least on the humoral response.

Please cite this article as: E. D. Mandon, A. Pizzorno, A. Traversier et al., Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.018

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Alternatively, CALX133ACE takes part to calixarene-based detergents/surfactants, which can establish hydrophobic interactions, salt bridges networks with basic residues, as well as p-stacking interactions with aromatic residues, and favors the solubilization and stabilization of very different types of membrane proteins, while preserving at its best their structural and functional integrity [16–20]. Previously, we successfully solubilized and purified influenza M2 ion channel from MDCK infected cells as a tetrameric and fully functional channel using a calixarene based detergent [18]. Likewise, we believe the current CALX133ACE-based split antigen preparation could contain more preserved and stabilized conformational antigenic epitopes that would contribute to generate a very efficient neutralizing antibody response, with a consequent improved protection against influenza infection. Another possible yet not exclusive explanation could also resides in the intrinsic composition of the CALX133ACE-based split preparation. Indeed, electron microscopy observations show mainly very large splitvirus particles and protein aggregates, whereas very few solubilized protein, like HA rosettes for example, are observed. Similarly, Tunable Resistive Pulse Sensing assay, crosslinking and native PAGE analyses suggest that CALX133ACE leads to the production of higher molecular weight species in comparison with Triton X100-based preparations. Among the different members of the calixarene-based detergent/surfactant family, CALX133ACE has low solubilization power due to its short aliphatic tail (3 carbons) [20]. Therefore, CALX133ACE may act as a ‘‘viral membrane splitter” instead of a more classical solubilizing detergent such as Triton X-100, hence leading to the production of larger membrane-embedded HA particles. In line with this hypothesis, Wei and colleagues have previously shown that large particulate antigens with repeated units can entail better immune responses than comparable amounts of monomeric antigens [32]. Moreover, it has been previously described that membrane-embedded envelop antigen proteins may induce efficient immunogenic responses per se, thanks to the potential role of the generated lipidic environment as natural immunogenic elicitor [33]. Also, the capacity of certain specific lipids to stabilize membrane protein oligomers has been reported [34]. Further studies are required to clearly determine whether some of the above mentioned hypotheses may explain such interesting properties of the CALX133ACE-based inactivated split influenza antigens. Likewise, monitoring of physical characteristics of higher molecular weight species within the CALX133ACE preparation and additional preclinical investigations are underscored. Most importantly, the absence of reactogenic responses with regard to larger membrane-embedded antigen particles needs to be demonstrated for further development of a CALX133ACE-based inactivated split influenza vaccine. Nevertheless, our preliminary stability data are promising, confirming comparable HA profiles between the CALX133ACE and Triton X-100-based H1N1 antigen splits for up to 6 months at 4 °C (Fig. S2A). As indicated in Fig. S2B, we also assessed the thermostability of both split preparations by performing 30-min incubations at different temperatures, ranging from 4 to 65 °C and observed that the CALX133ACE-based split presents comparable thermostability to the Triton X-100 split preparation, showing heat sensitivity at 65 °C.

8. Conclusions The CALX133ACE-based influenza inactivated split antigens developed in this study retain complete vaccine effectiveness at 10 to 30-fold lower equivalent HA doses than those of the commercial VaxigripÒ or FluviralÒ TIVs. Moreover, these CALX133ACE antigen preparations present a favorable preliminary safety/toxicity

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profile in vitro and in vivo models. In fact, CALX133ACE does not exhibit any toxicity alert in silico according to DEREK/EpiSuite analyses, neither mutagenic effect on Ames test, nor local toxicity was observed in mice or rabbits after intramuscular injection (data not shown). These results need to be confirmed in clinical safety studies. Finally, CALX133ACE exhibits long-term and heat stability comparable to that of the Triton X-100 split preparation. The CALX133ACE surfactant is of great public health and economic interest, since the need of a lower antigen dose per vaccine may translate into the production of more vaccine doses for the same amount of eggs used in comparison with the current process based on classical detergents. Beyond representing a realistic alternative to the potential new stringent regulations on the use of Triton X-100, the novel CALX133ACE, as well as the whole family of calixarene compounds, pave the way for future improvements of the vaccine manufacturing. Funding GB is the holder of the Canada Research Chair on Influenza Viruses and Other Respiratory Viruses (2006–13, renewed for 2013–20). Parts of this work were supported by INSERM, Université Claude Bernard Lyon 1 and Région Auvergne Rhône-alpes. Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [EDM, AT, ED and MRC are co-inventors of the patent entitled ‘‘Method for preparing a vaccine antigen, resulting vaccine antigen and uses (FR3025107A1, https://patents.google.com/patent/FR3025107A1/en).]. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2019.10.018. References [1] Pica N, Palese P. Toward a universal influenza virus vaccine: prospects and challenges. Annu Rev Med 2013;64:189–202. [2] http://www.who.int/news-room/fact-sheets/detail/influenza-(seasonal). [3] Graaf H, Faust SN. Fluarix quadrivalent vaccine for influenza. Expert Rev Vacc 2015;14(8):1055–63. [4] https://www.sanofipasteur.com/media/Project/One-Sanofi-Web/ sanofipasteur-com/en/media-room/docs/FactSheet_InfluenzaVaccines_EN.pdf. [5] McDonald J, Moore D. FluMist vaccine: Questions and answers - summary. Paediatr Child Health 2011;16(1):31. [6] Cox MM et al. Safety, efficacy, and immunogenicity of Flublok in the prevention of seasonal influenza in adults. Ther Adv Vacc 2015;3(4):97–108. [7] Manini I et al. Flucelvax (Optaflu) for seasonal influenza. Exp Rev Vacc 2015;14 (6):789–804. [8] Krammer F, Palese P. Advances in the development of influenza virus vaccines. Nat Rev Drug Discov 2015;14(3):167–82. [9] Soema PC et al. Current and next generation influenza vaccines: Formulation and production strategies. Eur J Pharm Biopharm 2015;94:251–63. [10] Belanger JM et al. Orthogonal inactivation of influenza and the creation of detergent resistant viral aggregates: towards a novel vaccine strategy. Virol J 2012;9:72. [11] Gerdil C. The annual production cycle for influenza vaccine. Vaccine 2003;21 (16):1776–9. [12] Grohskopf LA et al. Prevention and control of seasonal influenza with vaccines: recommendations of the Advisory Committee on Immunization PracticesUnited States, 2017–18 Influenza Season. Am J Transplant 2017;17 (11):2970–82. [13] https://ecdc.europa.eu/en/seasonal-influenza/prevention-and-control/ seasonal-influenza-vaccines. [14] Scheiblhofer S et al. Influence of protein fold stability on immunogenicity and its implications for vaccine design. Exp Rev Vacc 2017;16(5):479–89. [15] Hardy D et al. The yin and yang of solubilization and stabilization for wild-type and full-length membrane protein. Methods 2018.

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E.D. Mandon et al. / Vaccine xxx (xxxx) xxx

[16] Agez M et al. Molecular architecture of potassium chloride co-transporter KCC2. Sci Rep 2017;7(1):16452. [17] Desuzinges Mandon E et al. Novel systematic detergent screening method for membrane proteins solubilization. Anal Biochem 2017;517:40–9. [18] Desuzinges Mandon E et al. Expression and purification of native and functional influenza A virus matrix 2 proton selective ion channel. Protein Expr Purif 2017;131:42–50. [19] Rosati A et al. BAG3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages. Nat Commun 2015;6:8695. [20] Matar-Merheb R et al. Structuring detergents for extracting and stabilizing functional membrane proteins. PLoS ONE 2011;6(3):e18036. [21] http://www.medtecheurope.org/node/1060. [22] Wood JM et al. An improved single-radial-immunodiffusion technique for the assay of influenza haemagglutinin antigen: application for potency determinations of inactivated whole virus and subunit vaccines. J Biol Stand 1977;5(3):237–47. [23] Kon TC et al. Influenza vaccine manufacturing: effect of inactivation, splitting and site of manufacturing. Comparison of influenza vaccine production processes. PLoS ONE 2016;11(3):e0150700. [24] World Health Organization. Global Influenza Surveillance Network Manual for the laboratory diagnosis and virological surveillance of influenza; 2011. http:// www.who.int/influenza/gisrs_laboratory/manual_diagnosis_surveillance_ influenza/en/. [25] Chaptal V et al. Quantification of detergents complexed with membrane proteins. Sci Rep 2017;7:41751.

[26] Fortin T et al. Clinical quantitation of prostate-specific antigen biomarker in the low nanogram/milliliter range by conventional bore liquid chromatography-tandem mass spectrometry (multiple reaction monitoring) coupling and correlation with ELISA tests. Mol Cell Proteom 2009;8 (5):1006–15. [27] Akpinar F, Yin J. Characterization of vesicular stomatitis virus populations by tunable resistive pulse sensing. J Virol Methods 2015;218:71–6. [28] Hirst GK. The quantitative determination of influenza virus and antibodies by means of red cell agglutination. J Exp Med 1942;75(1):49–64. [29] Lee KK et al. Quantitative determination of the surfactant-induced split ratio of influenza virus by fluorescence spectroscopy. Hum Vaccin Immunother 2016;12(7):1757–65. [30] Hendriks J et al. An international technology platform for influenza vaccines. Vaccine 2011;29(Suppl 1):A8–A11. [31] Wibowo N et al. Protective efficacy of a bacterially produced modular capsomere presenting M2e from influenza: extending the potential of broadly cross-protecting epitopes. Vaccine 2014;32(29):3651–5. [32] Wei CJ et al. Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza virus. J Virol 2008;82(13):6200–8. [33] Gregoriadis G. Immunological adjuvants: a role for liposomes. Immunol Today 1990;11(3):89–97. [34] Gupta K et al. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature 2017;541(7637):421–4.

Please cite this article as: E. D. Mandon, A. Pizzorno, A. Traversier et al., Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.018