Introduction, synopsis of results and conclusions

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the 2019 coronavirus disease (COVID-19) pandemic which presents an unprecedented challenge to societies globally. Effective vaccines and sensitive diagnostic tools for COVID-19 are urgently needed, and systems to produce and deliver these tools in sufficient quantities are required. As the SARS-CoV-2 trimeric Spike complex is a major target of the immune system, it could be a useful ingredient for vaccine and diagnostic applications. Stabilized trimeric Spike proteins have been selected as antigen of choice for RNA and virus-vector based vaccine candidates that are currently under development (Moderna, Novavax, Pfizer, J&J etc.). The efficacy of these vaccines was recently reported to be as high as 95 %. However, to maintain long-term protective immunity, follow-up vaccinations may be required. An adjuvated, subunit vaccine based on the stabilized trimeric Spike protein could be ideal for this purpose. Subunit vaccines can be produced in a cost-effective way and can be transported and stored lyophilized at ambient temperature. Also, whether any vaccination approach has elicited a sufficiently protective immune response against SARS-CoV-2 and the perpetuity of the immune response needs to be verified. For this reason, there will be a high demand for diagnostic tests for quantification of SARS-CoV-2-specific antibodies. The antigen which provides the best sensitivity and specificity for detection of SARS-CoV-2 antibodies is the trimeric form of the Spike protein a conclusion enforced by here presented data.

The viral Spike (S) protein complex is a surface-exposed homo-trimeric structure that mediates entry into host cells. The Spike engages the cellular host receptor ACE2 and allows virus-host membrane fusion. The Spike complex is considered the primary target of neutralizing antibodies. The ideal diagnostic test for SARS-CoV-2 antibodies would detect all antibodies directed against the trimeric Spike protein complex. Production of such diagnostic tests requires the production of large quantities of highly purified S protein in its natural prefusion conformation.

In this study, we developed a Chinese Hamster Ovary (CHO) cell based production process that yields high-quality trimeric Spike protein. This protein can be used for diagnostic tests as well as for vaccine applications. We used the fully characterized and CMC-compliant CHOExpress™ cell host (ExcellGene SA), single use equipment, chemically defined media and additives and obtained high-quality product at high volumetric productivity. This process can be readily scaled up to produce kilograms of trimeric Spike protein, should the demand rise to such a level. We showed that the purified trimeric Spike is efficient in blocking virus entry into cells when provided in molar excess to cultures infected with SARS-CoV-2. We also demonstrated the highest thus far seen sensitivity and specificity of Spike proteins for detection of Covid-19 patient-derived sera. We have modeled the product based on EM- images with both the “Wuhan” strain, as well as the D614G variant of the spike protein.

Using an optimized CHO expression system, and a scalable, chemically defined production process, trimeric SARS CoV-2 Spike proteins with mammalian-type glycosylation could be provided. The protein is of high purity and trimeric. Functional analysis showed it to be efficient in blocking virus infectivity in an in-vitro model. In addition diagnostic performance of the trimeric Spike for SARS-CoV-2 specific IgG was shown to exceed that of RDB and monomeric S1 protein.

Results

Design and selection of CHO manufacturable SARS-CoV-2 trimeric spikes

In order to define the optimal construct design to produce a soluble trimeric Spike protein we evaluated the expression with various S protein-variants (Figure 1) by CHO transient expression. The transmembrane domain and the C terminal intracellular tail were removed and replaced by a T4 foldon DNA sequence with or without (GGGS)n linker and a 8xHis tag encoding DNA. The furin cleavage site RRAR is mutated to be non-functional. WT Spike sequences of both the Wuhan strain and the D614G strain and amino-acid changes K986P/V987P (“2P”) for locking the protein into a prefusion conformation were also used. In addition, a receptor binding domain truncation of the trimeric Spike was used (RBD-His). S proteins variants with a scrambled furin cleavage site were found better expressed. Mutation of the furin cleavage site seemed to increase trimer assembly and/or stability, suggesting that trimer assembly via T4 foldon occurs in the Golgi (Trans-Golgi network where furin is active). The same construct modifications were also applied for the D614G mutation in the Spike protein which has recently replaced to a large extent the Wuhan version.

The version named Spike_ΔCter_ΔFurin_2P_T4_His showed highest expression and appeared to be most promising in terms of trimeric assembly.

CHO expression and purification of SARS-CoV-2 proteins

Stable recombinant cell pools expressing trimeric Spike and RBD were generated using puromycin selection. From a total of 300 clonally derived cell populations, those with the highest expression levels of trimeric Spike and RBD were expanded and a fed-batch production process was developed. Optimal conditions were selected on the basis of product yield, product quality, viable cell density (VCD) and cell viability. The production process was scaled up to 200 mL shake flasks, to 10 L and to 40 L stirred tank bioreactors (STR). Viable cell density (VCD) and viability remained high for at least 10 days. From the authors’ experience in developing high-yielding manufacturing processes for clinically relevant recombinant antibodies and other proteins since almost 20 years, there is high confidence to assure feasibility to any scale of operation, up and beyond the 2,000 Liter scale.

Molecular characterization and electron microscopy analysis of trimeric Spikes

Trimeric Spike and RBD from cell-free culture supernatant were purified using an immobilized metal affinity chromatography (IMAC) capture step followed by preparative size exclusion. Eluates were concentrated and formulated at 1 mg/mL in PBS pH 7.4. Purity of the final products was estimated to be over 95 % as analyzed by HPLC-SEC (Figure 2 a and b).

HPLC-SEC (non-denaturing conditions) indicates that the S protein complex is trimeric (about 460 kDa) and the RBD is monomeric. The expected size of 150-160 kDa and 29 kDa for the Spike and RBD monomers, respectively, were confirmed by reducing SDS-PAGE (Figure 2 c, d). The heavy glycosylation of trimeric Spike contributes significantly to size heterogeneity, as has been seen with other CHO cell produced virus derived proteins, such as truncated surface proteins from Ebola and HIV. Trimeric confirmation was also confirmed using negative-stain electron microscopy and 3D modeling (Figure 2 d - g).

Inhibition of SARS-CoV-2 infection by SARS-CoV-2 proteins

SARS-CoV-2 virus was mixed with serially diluted RBD or trimeric Spike and the mixture was inoculated on Vero E6 cells. 48 hours later, SARS-CoV-2 infection was visualized and quantified (Figure 3 a and b). The presence of both the RBD Spike fragment and trimeric Spike reduced infection of Vero E6 cells. For RBD, reduction of infectivity was observed at a concentration of 20 µM and higher. For the trimeric Spike (both for the Wuhan variant and the D614G variant), a near 100-fold lower concentration reduced infection (Figure 3 b). Taken together, these results show that CHO-produced trimeric Spike and RBD are able to compete with SARS-CoV-2 for binding to host cells. No differences were observed between the 614D (Wuhan) and the D614G variants of the trimeric Spike.

Sensitivity and specificity of SARS CoV-2 Spike proteins for detection of antibodies

The trimeric Spike and RBD proteins were used to detect SARS-CoV-2–specific IgG in a semi-quantitative bead-based serological assay. We specifically selected a set of COVID-19 serum samples that cover a large range of SARS-CoV-2–specific antibody concentrations. Sera (n = 72) were collected from PCR-confirmed non-hospitalized (n = 63) and hospitalized (n = 9) COVID-19 patients between days 4 and 40 after disease onset. Control sera (n = 79) were collected before the emergence of SARS-CoV-2 from patients with seasonal corona-induced Influenza-like illness (n = 44), non-corona, Influenza-like illness (n = 29), and from healthy individuals (n = 6).

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