2016 [Methods in Molecular Biology] Vaccine Design Volume 1403 __ Development of a SARS Coronavirus Vaccine from Recombi

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 ?TM) that was expressed using a baculovirus insect cell expression Clifton McPherson et al. 271 platform. As the S ?TM 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 ?TM 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 ?TM. 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 expresSF+ ® insect cells with the recombinant baculovirus, the S ?TM protein is expressed and secreted into the cell culture medium [ 17]. The procedure detailed below for purifying the SARS S ?TM 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 ?TM 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 ?TM 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 ?TM 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 ?m fi lter, and the purifi ed protein is stored at ?20 °C. Prior to intended use, the purifi ed SARS S ?TM 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 (6000xg 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 µm Filtration Fig. 1 Process fl ow diagram of SARS S ?TM 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 ?m 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-?- 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 ?m 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 ?m Nylon (Falcon). 15. RPMI complete medium with 10 % heat-inactivated FBS (Invitrogen Life Technologies). 16. Red blood cell (RBC) lysis buffer (155 mM NH 4Cl; 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-?, 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 ?m 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 ?500 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 ?500 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 ?500 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 ?10 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-?- 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-?- 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-?- 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-?- 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 ?TM protein. 12. Pool fractions containing detectable SARS S ?TM 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 ?m 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 ?TM protein. 11. Retentate line should be connected to process tank, and valve should be open. 12. Allow S ?TM to recirculate through the system at a fl ow rate of ?200 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 ?200 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 ?m fi lter into a sterile Nalgene polycarbonate bottle. This is the S ?TM bulk drug substance. 27. Remove aliquots for testing. 28. Store bulk drug substance at ?20 °C. 1. Testing and acceptance criteria for SARS S ?TM 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 ?g 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 ?TM bulk drug sub- stance in a laminar fl ow hood by aseptic simple admixture of the Advax™ suspension with the S ?TM 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 twice 3 weeks apart by an intramuscular injection into the thigh, in order to mimic the most common route of human vaccine administration. The maximum volume that can be injected into an adult mouse thigh muscle is 50 ?l. If the vaccine cannot be reduced to this volume, then provid- ing the vaccine is not reactogenic and then larger vaccine vol- umes can be administered by injection of 50 ?l amounts into multiple legs. 3. Starting at 1 week post-immunization, at intervals of 1–4 weeks mice are bled using cheek vein bleeding using a lancet in order to obtain ~25–50 ?l of blood from which serum is 3.7 Animal Immunogenicity Testing Table 1 SARS S ?TM analytical tests and acceptance criteria Parameter Method Acceptance criterion Identity SDS-PAGE/ Western blot Detection of approx. 150 kDa protein with SARS S antiserum DNA content PicoGreen ?15 ng/dose pH USP <791> 7.0 ± 0.4 Osmolality USP <785> ?330 mOsm/hg Bacterial endotoxin USP <85> <10 EU/dose Purity SDS-PAGE/ Western blot ?90 % Lentil lectin content SDS-PAGE/ Western blot <10 ng lentil lectin/?g SARS S ?TM Microbial limits USP <61> <10 CFU/mL Total protein Bicinchoninic acid assay Perform and report Potency ELISA ?60 % of total protein content Clifton McPherson et al.