【病毒外文文獻】2016 [Methods in Molecular Biology] Vaccine Design Volume 1403 __ Development of a SARS Coronavirus Vaccine from Recombi

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269 Sunil Thomas ed Vaccine Design Methods and Protocols Volume 1 Vaccines for Human Diseases Methods in Molecular Biology vol 1403 DOI 10 1007 978 1 4939 3387 7 14 Springer Science Business Media New York 2016 Chapter 14 Development of a SARS Coronavirus Vaccine from Recombinant Spike Protein Plus Delta Inulin Adjuvant Clifton McPherson Richard Chubet Kathy Holtz Yoshikazu Honda Okubo Dale Barnard Manon Cox and Nikolai Petrovsky Abstract Given periodic outbreaks of fatal human infections caused by coronaviruses development of an optimal coronavirus vaccine platform capable of rapid production is an ongoing priority This chapter describes the use of an insect cell expression system for rapid production of a recombinant vaccine against severe acute respiratory syndrome coronavirus SARS Detailed methods are presented for expression purifi cation and release testing of SARS recombinant spike protein antigen followed by adjuvant formulation and animal testing The methods herein described for rapid development of a highly protective SARS vaccine are equally suited to rapid development of vaccines against other fatal human coronavirus infections e g the MERS coronavirus Key words Coronavirus SARS MERS Vaccine Adjuvant Delta inulin Advax adjuvant Baculovirus Manufacture cGMP 1 Introduction The severe acute respiratory syndrome coronavirus SARS was fi rst identifi ed in 2003 after a series of fatal pneumonia cases char acterized by an infl ammatory cell infi ltrate with diffuse alveolar damage 1 started in Hong Kong before spreading to other coun tries 2 Before being controlled by quarantine measures 8000 humans were infected with fatality as high as 50 in the elderly and an overall case fatality rate of 10 2 More recently the Middle East respiratory syndrome coronavirus MERS has caused a series of serious and in some cases fatal human infections 3 Given the risk of future serious human coronavirus outbreaks development of a suitable vaccine platform to protect against such viruses is a major priority These vaccines present several challenges including the rapidity with which these outbreaks develop and hence the need for rapid vaccine manufacture Hence a successful coronavirus vaccine platform must overcome multiple challenges 270 SARS CoV is a positive stranded RNA virus 29 7 kb in length with 14 open reading frames 4 Initial SARS vaccine candidates were produced from inactivated virus Inactivated whole virus vac cines provided only modest protection inducing low neutralizing antibody titers that did not protect against infection but were asso ciated with faster lung clearance of virus 5 However immuniza tion of mice with inactivated vaccines either alone or formulated with alum adjuvant resulted in severe lung eosinophilic pathology in response to virus challenge 6 9 similar to enhanced lung pathology seen with SARS virus reexposure after primary infection 10 Hence a major challenge when developing a SARS vaccine is to identify strategies to avoid lung eosinophilic pathology A further challenge when developing vaccines based on inacti vated SARS virus is the need for high containment biosafety level 3 cGMP manufacturing facilities 11 This makes vaccine manu facture more complex and expensive and restricts the number of vaccine doses that can be rapidly manufactured To counter these challenges it would be preferable to produce a subunit vaccine that just like inactivated virus was able to induce neutralizing anti bodies against SARS CoV but rather than requiring BSL3 manu facture was able to be produced in a regular recombinant protein manufacturing environment The potential solution to this problem lies in the coronavirus spike protein S protein which in the case of SARS virus binds to angiotensin converting enzyme 2 and CD209L and induces receptor mediated virus endocytosis thereby being critical to virus entry into target cells 12 13 S protein could thereby provide an ideal antigen with which to induce neutralizing antibodies against SARS virus 14 15 However while immunization with S protein in animal models provided some protection against SARS virus 16 17 S protein immunized animals when subsequently chal lenged with live virus developed severe lung eosinophilic immuno pathology with this problem exacerbated even further when S protein vaccine was formulated with alum adjuvant 6 9 A simi lar problem of lung eosinophilic immunopathology has been seen with other viruses including respiratory syncytial virus RSV vac cines which prime for an excessive and harmful Th2 dominated lung immune response upon subsequent virus exposure 18 Lung eosinophilic immunopathology is exacerbated by formula tion of such vaccines with any adjuvant that induces excess Th2 immune polarization e g aluminum salt adjuvants 6 9 Hence while S protein would appear to be an ideal SARS vaccine antigen there is fi rst the need to reduce the risk of lung eosinophilic immu nopathology being induced by the vaccine Described below are methods used for the development and manufacture of a recombinant subunit vaccine based on an S pro tein antigen lacking transmembrane and cytoplasmic domains S uni0394TM that was expressed using a baculovirus insect cell expression Clifton McPherson et al 271 platform As the S uni0394TM protein antigen itself has low immunoge nicity 16 17 steps are also described for formulation with a safe and effective adjuvant 19 As aluminum adjuvants are contraindi cated for SARS vaccines given they may exacerbate lung eosino philic pathology instead methods are described for formulation of the S uni0394TM protein with Advax a safe and effective adjuvant based on delta inulin 20 21 that has been previously shown in animal models to enhance the immunogenicity of a broad range of viral and bacterial antigens 22 30 and has also been shown safe and effective in preliminary human clinical trials 31 32 Notably Advax adjuvant was recently shown to enhance the immunogenic ity and protection conferred by both inactivated and recombinant SARS vaccines without the excess Th2 bias of alum adjuvants and hence without the risk of inducing lung eosinophilic immunopa thology 33 For this project a recombinant baculovirus was designed to express the ectodomain of the SARS S protein lacking the trans membrane and cytoplasmic domains 17 The recombinant pro tein expressed by this vector was termed SARS S uni0394TM This truncated version of the S protein was selected as it contains the receptor binding domain RBD and was able to be expressed in insect cells at a higher level than the full length membrane bound version The SARS S RBD has been shown to induce neutralizing antibodies against the SARS CoV 14 15 After infection of expres SF insect cells with the recombinant baculovirus the S uni0394TM protein is expressed and secreted into the cell culture medium 17 The procedure detailed below for purifying the SARS S uni0394TM protein can be followed after production of the protein in a bacu lovirus insect cell system The process is designed for a 45 L fer mentation in a 60 L bioreactor but may be adjusted to other scales as necessary Following harvest the SARS S uni0394TM protein is puri fi ed by column chromatography a schematic is provided in Fig 1 In the fi rst step the supernatant is fl owed through linked UNOsphere S UNO S and DEAE sepharose columns DNA and protein impurities are removed and the SARS S uni0394TM protein remains in the fl ow through The fl ow through from the UNO S DEAE step is applied to lentil lectin sepharose SARS S uni0394TM binds and impurities are removed Following elution from the len til lectin sepharose column the protein is concentrated and buffer is exchanged by ultrafi ltration The retentate from the ultrafi ltra tion step is processed through a 0 22 uni03BCm fi lter and the purifi ed protein is stored at 20 C Prior to intended use the purifi ed SARS S uni0394TM protein is mixed under conditions with a suspension of Advax delta inulin adjuvant particles and either aseptically fi lled into single dose vials or loaded directly into syringes ready for vaccination Lastly the adjuvanted SARS vaccine is tested for effi cacy and safety in animal immunogenicity and SARS challenge models SARS Vaccine Development 272 Fermentation in serum free media Centrifugation Cells harvested by low speed centrifugation 6000 xg 15 minutes SARS S TM protein recovered in supernatant Pellet discarded Cellular material removed Initial Purification Chromatography Ion Exchange UNO S DEAE linked columns Supernatant is applied to the columns Equilibration Buffer 1 0 5M Tris pH 7 4 Equilibration Buffer 2 20 mM Tris pH 7 4 SARS S TM protein flows through the columns The linked columns are washed to baseline with 20 mM Tris Contaminants discarded Additional viral clearance DNA and protein impurities reduced Final Purification Lentil Lectin Chromatography SARS S TM protein FT from linked UNO S DEAE is applied to lentil lectin column Equilibration Buffer Wash buffer 20 mM Tris pH 7 4 A Step Elution was performed with varying conc of N Methyl D mannopyranoside Elution buffer 50 mM 100mM 200mM 500 mM N Methyl D mannopyranoside in 20mM Tris pH 7 4 SARS S TM protein binds to the lentil lectin column The column is washed with Equilibration Wash buffer then TM S protein is eluted with Elution buffer Product is pooled based on level of purity Protein impurities removed Diafiltration and Concentration Lentil lectin column eluate are pooled and concentrated using a Sartocon Slice Benchtop unit equipped with a 0 1m 2 50 kDa MWCO Sartocon Slice cassette Diafiltration Buffer 150mM NaCl 2 5mM SodiumPhosphate pH 6 8 7 2 in WFI Buffer Exchange Buffer is exchanged 10 times with diafiltration buffer Retentate is concentrated to a target volume of 500 mLs Protein is concentrated and buffer is exchanged Small protein contaminants smaller than about 50 kDa are removed in the filtrate 0 2 m Filtration Fig 1 Process fl ow diagram of SARS S uni2206TM protein Clifton McPherson et al 273 2 Materials 1 1 L Nalgene centrifuge bottles Thermo Scientifi c 2 High speed centrifuge 3 Centrifuge rotor to accommodate 1 L bottles 4 Sterile 50 L Nalgene carboy Thermo Scientifi c 5 0 22 uni03BCm fi lter EMD Millipore 1 Two BPG columns GE Healthcare 2 UNOsphere S chromatography resin Bio Rad 3 DEAE Sepharose Fast Flow chromatography resin GE Healthcare 4 Chromatography system equipped with UV and conductivity monitors 5 Tris hydroxymethyl aminomethane Trizma Sigma Aldrich 6 Concentrated HCl 7 Purifi ed water 8 Sterile 50 L Nalgene carboy Thermo Scientifi c 1 XK 50 column GE Healthcare 2 Lentil lectin Sepharose 4B GE Healthcare 3 Chromatography system equipped with UV and conductivity monitors 4 Tris hydroxymethyl aminomethane Sigma Aldrich 5 Concentrated HCl 6 Purifi ed water 7 N methyl uni03B1 D mannopyranoside Sigma Aldrich 8 Sterile Nalgene square 250 mL polycarbonate bottles Thermo Scientifi c 1 Sartocon Slice 200 bench top system Sartorius 2 Sartocon Slice 200 PESU cassette Sartorius 3 Pump 4 Sodium phosphate monobasic monohydrate Sigma Aldrich 5 Sodium phosphate dibasic 12 hydrate Sigma Aldrich 6 Sodium chloride Sigma Aldrich 7 Purifi ed water 8 Pipettes 9 Microcentrifuge tubes 10 Sterile Nalgene square 500 mL polycarbonate bottle Thermo Scientifi c 2 1 Fermentation Harvest 2 2 UNO S DEAE Column Chromatography 2 3 Lentil Lectin Capture Chromatography 2 4 Concentration Ultrafi ltration and 0 22 uni03BCm Filtration SARS Vaccine Development 274 1 Delta inulin adjuvant suspended in bicarbonate buffer Vaxine Pty Ltd 2 CpG oligonucleotide powder Oligo Factory USA 3 Water for injection Baxter 1 Female 6 8 week old BALB c mice weighing 18 20 g 2 0 5 mL Insulin syringes BD 3 5 mL Syringes 4 25G 5 8 needles 5 Animal lancet 4 mm Medipoint Inc USA 6 96 Well ELISA plates Greiner Bio One 7 24 Well culture plates Greiner Bio One 8 0 1 M Sodium carbonate buffer pH 9 6 9 1 BSA PBS 10 Biotinylated anti mouse IgG IgG1 IgG2a IgG2b IgG3 or IgM antibodies Abcam 11 Streptavidin HRP BD Biosciences 12 TMB substrate KPL USA 13 1 M Phosphoric Acid 14 Cell strainers 70 uni03BCm Nylon Falcon 15 RPMI complete medium with 10 heat inactivated FBS Invitrogen Life Technologies 16 Red blood cell RBC lysis buffer 155 mM NH 4 Cl 10 mM KHCO 3 0 1 mM EDTA pH 7 3 17 Carboxy fl uorescein diacetate succinimidyl ester CFSE Invitrogen Life Technologies 18 MultiScreen HTS 96 well fi ltration plate Merck Millipore 19 Anti mouse CD16 CD32 BD Biosciences 20 Anti mouse CD4 APC BD Biosciences 21 Anti mouse CD8a PE Cy7 BD Biosciences 22 Anti mouse IFN uni03B3 IL 2 IL 4 antibody pairs BD Biosciences 23 LEAF anti mouse IL 17A and biotin anti mouse IL 17A anti body BioLegend USA 1 Female 4 8 week old BALB c mice weighing 18 20 g 2 SARS CoV virus strain Urbani 200300592 Centers for Disease Control and Prevention Atlanta GA USA 3 Vero 76 cell line American Type Culture Collection Manassas VA USA 4 Hematoxylin and eosin stain 2 5 Adjuvant Formulation 2 6 Mouse Immunogenicity Testing 2 7 Animal Challenge Studies Clifton McPherson et al 275 5 Rat monoclonal antibody Clone MT 14 7 to eosinophil major basic protein MBP Lee Laboratory Mayo Clinic Arizona 6 DAB chromogen 3 Methods 1 Separate cells and culture supernatant by centrifugation at 5900 g at 2 8 C for 15 min 2 Transfer culture supernatant to a sterile 50 L carboy 3 After all culture supernatant is collected fi lter through 0 22 uni03BCm fi lter into a second sterile 50 L carboy see Note 1 4 Store at 2 8 C 1 Pack a BPG column with 1 9 L of UNO S resin see Note 2 2 Pack a BPG column with 1 5 L of DEAE Sepharose Fast Flow resin 3 pH equilibrate the UNO S column with 0 5 M Tris pH 7 4 at a fl ow rate of 200 400 mL min until outfl ow pH is 7 0 7 7 This step typically requires 3 column volumes of buffer see Note 3 4 Continue equilibration of the UNO S column with 20 mM Tris pH 7 4 at a fl ow rate of 200 400 mL min until outfl ow is pH 7 2 7 5 and conductivity is uni2264500 mS This step typically requires 5 column volumes 5 pH equilibrate the DEAE column with 0 5 M Tris pH 7 4 at a fl ow rate of 200 400 mL min until outfl ow pH is 6 9 7 6 This step typically requires 3 column volumes of buffer 6 Continue equilibration of the DEAE column with 20 mM Tris pH 7 4 at a fl ow rate of 200 400 mL min until outfl ow is pH 7 0 7 5 and conductivity is uni2264500 mS This step typically requires 5 column volumes 7 Connect the DEAE column to the outfl ow of the UNO S column 8 Equilibrate the linked UNO S and DEAE columns with 20 mM Tris pH 74 at a fl ow rate of 200 400 mL min until outfl ow is pH 6 9 7 6 and conductivity is uni2264500 mS 9 Apply the culture supernatant from Subheading 2 1 to the linked UNO S DEAE columns at a fl ow rate of not more than 300 mL min see Note 4 10 Begin collecting the fl ow through from the linked columns into a 50 L carboy when the UV trace begins to rise 11 Wash the linked columns with 20 mM Tris pH 7 4 and collect the wash in the carboy with the column fl ow through 3 1 Fermentation Harvest 3 2 UNO S DEAE Column Chromatography SARS Vaccine Development 276 12 Collect uni226410 L of wash and stop collection of the wash when the UV trace returns to baseline 13 Proceed to lentil lectin capture chromatography 1 Pack XK 50 column with 250 mL of lentil lectin sepharose see Note 2 2 Equilibrate lentil lectin column with 20 mM Tris pH 7 4 at a fl ow rate of 50 100 mL min using 10 column volumes of buffer pH should be 7 2 7 5 3 Load UNO S DEAE fl ow through to the lentil lectin column at a fl ow rate of 50 100 mL min see Note 5 4 Wash column with 20 mM Tris pH 7 4 at a fl ow rate of 50 100 mL min for 5 column volumes or until UV trace returns to baseline 5 Collect fractions in sterile Nalgene square 250 mL polycarbon ate bottles 6 Elute with 2 column volumes of 50 mM N methyl uni03B1 D mannopyranoside and 20 mM Tris pH 7 4 at a fl ow rate of 50 100 mL min Collect two 250 mL fractions 7 Elute with 2 column volumes of 100 mM N methyl uni03B1 D mannopyranoside and 20 mM Tris pH 7 4 at a fl ow rate of 50 100 mL min Collect two 250 mL fractions 8 Elute with 2 column volumes of 200 mM N methyl uni03B1 D mannopyranoside and 20 mM Tris pH 7 4 at a fl ow rate of 50 100 mL min Collect two 250 mL fractions 9 Elute with 5 column volumes of 500 mM N methyl uni03B1 D mannopyranoside and 20 mM Tris pH 7 4 at a fl ow rate of 50 100 mL min Collect 250 mL fractions until UV trace is fl at and stable 10 Store fractions at 2 8 C 11 Analyze all fractions by SDS PAGE and Western blot to deter mine which fractions contain SARS S uni0394TM protein 12 Pool fractions containing detectable SARS S uni0394TM protein 1 Assemble Sartocon Slice 200 bench top ultrafi ltration system with a 0 1 m 2 50 kDa molecular weight cutoff Sartocon Slice cassette according to the manufacturer s instructions 2 Attach a process tank according to the manufacturer s instructions 3 Fill process tank with water for injection WFI 4 Close permeate valve and circulate WFI through the system at 200 mL min for 3 4 min 5 Open permeate valve 6 Increase circulation rate to 600 800 mL min 3 3 Lentil Lectin Capture Chromatography 3 4 Concentration Ultrafi ltration and 0 22 uni03BCm Filtration Clifton McPherson et al 277 7 Adjust retentate valve to obtain a transmembrane pressure TMP of 8 0 17 0 psi 8 Continue rinsing for 5 10 min 9 Repeat steps 3 8 using diafi ltration buffer 150 mM NaCl 2 5 mM NaPO 4 pH 6 8 7 2 in WFI 10 Fill process tank with pooled SARS S uni0394TM protein 11 Retentate line should be connected to process tank and valve should be open 12 Allow S uni0394TM to recirculate through the system at a fl ow rate of uni2264200 mL min for 3 4 min 13 Increase recirculation fl ow rate to 600 800 mL min 14 Ensure a TMP of 13 0 17 0 psi 15 Monitor volume in process tank 16 Stop concentration when volume in process tank is approxi mately 400 mL This is the initial concentration retentate 17 Set up a 2 L vessel to siphon into process tank 18 Fill container with 2 L of diafi ltration buffer 150 mM NaCl 2 5 mM NaPO 4 pH 6 8 7 2 in WFI 19 Retentate valve should be open 20 Start pump and circulate at a fl ow rate of uni2264200 mL min for 3 4 min 21 Increase fl ow rate to 600 800 mL min 22 Adjust retentate valve to maintain a TMP of 13 0 17 0 psi 23 Monitor volume of diafi ltration buffer in vessel 24 Continue until a total volume of diafi ltration buffer equal to 10 times the volume of initial concentration retentate has been used 25 Volume in process tank should be approximately equal to ini tial concentration retentate volume This is the diafi ltration retentate 26 Process the diafi ltration retentate through a 0 22 uni03BCm fi lter into a sterile Nalgene polycarbonate bottle This is the S uni0394TM bulk drug substance 27 Remove aliquots for testing 28 Store bulk drug substance at 20 C 1 Testing and acceptance criteria for SARS S uni0394TM are listed in Table 1 1 Advax is a preservative free sterile suspension of delta inulin microparticles at 50 mg mL in a bicarbonate buffer which when combined with vaccine antigen enhances both Th1 and Th2 immunity in a balanced fashion 3 5 Antigen Release Testing 3 6 Vaccine Adjuvant Formulation SARS Vaccine Development 278 2 To further enhance Th1 and reduce Th2 immune bias 10 uni03BCg CpG oligonucleotide per 1 mg delta inulin is added to the Advax adjuvant as a simple admixture 3 Advax adjuvant formulations are administered to mice at a standardized dose of 1 mg delta inulin per mouse irrespective of the antigen dose 4 Advax adjuvant is formulated with S uni0394TM bulk drug sub stance in a laminar fl ow hood by aseptic simple admixture of the Advax suspension with the S uni0394TM bulk drug substance and drawing up the combined milky white suspension into a 0 5 mL insulin syringe immediately prior to immunization 1 Vaccine immunogenicity studies can be conveniently performed on adult female BALB c mice at 6 8 weeks of age but can also be performed on other strains such as C57BL 6 see Note 6 2 Mice are immunized