WO2022061005A1

WO2022061005A1 - Methods of design and production of sars-cov-2 recombinant antigens in bacteria and their use in serology tests

Info

Publication number: WO2022061005A1
Application number: PCT/US2021/050702
Authority: WO
Inventors: Maria KIREEVA; Gabriel FITZGERALD; Andrei Komarov; Anna KAZNADZEY
Original assignee: Virintel, Llc
Priority date: 2020-09-16
Filing date: 2021-09-16
Publication date: 2022-03-24

Abstract

This invention relates in one aspect to methods for producing recombinant antigens in bacterial cells. The methods may comprise the following steps: growing bacterial cells which express a recombinant SARS-CoV-2 surface glycoprotein fragment fused to a purification tag, inducing expression of the fragment, harvesting the cells, resuspending the cells in a lysis buffer and sonicating this lysis mixture; and purifying and refolding the antigen from the lysis mixture. In another aspect, this invention relates to antigens for serological tests that detect SARS-CoV-2 antibodies in a subject's sample.

Description

METHODS OF DESIGN AND PRODUCTION OF SARS-CoV-2 RECOMBINANT ANTIGENS IN BACTERIA AND THEIR USE IN SEROLOGY TESTS

TECHNICAL FIELD

[0001] This invention relates to methods for producing recombinant antigens which can be used as reagents in different methods, including serological tests that detect if SARS-CoV-2 antibodies are present in a subject's sample. The subject samples are biological fluids including, but not limited to blood serum, plasma, whole blood, including dried blood spot, saliva, and mucus collected by nasal, throat, and nasopharyngeal swabs.

BACKGROUND

[0002] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the strain of coronavirus that causes a respiratory illness known as coronavirus disease 2019 (COVID-19).

[0003] Efficient public health response to the developing COVID-19 pandemic requires development of diagnostic tools for revealing seroconversion (immune response) to SARS-CoV-2 infection. Sensitive and specific antibody tests are essential for epidemiological and public health studies. They can be used to establish the extent of an outbreak, map its overall geographical distribution and the hotspot locations, and identify groups that are at higher risk of infection. This information serves as a foundation for public health measures and control strategies (Peeling et al., 2020). Vaccine development, testing, and validation is another important area of serology test's application (Okba et al., 2020, Nie et al., 2020).

[0004] Antigen selection is crucial for development of an effective serological test aiming at rapid and accurate detection of SARS-CoV-2 antibodies in a subject's sample.

[0005] Currently, there is a need in the field for recombinant antigens and methods by which the antigens can be produced.

SUMMARY [0006] In one aspect, the present disclosure provides a method for producing an antigen for serological tests that detect SARS-CoV-2 antibodies, the method comprising: a) growing bacterial cells which express a recombinant SARS-CoV-2 surface glycoprotein fragment fused to a purification tag, b) inducing expression of the fragment, c) harvesting the cells, d) resuspending the cells in a lysis buffer and sonicating this lysis mixture; and e) purifying and refolding the antigen from the lysis mixture.

[0007] In some preferred embodiments of the method, the fragment may be a peptide with SEQ ID NO. 1 or a peptide with at least 80% amino acid sequence identity to the peptide with SEQ ID NO. 1 .

[0008] In some embodiments of the method, purification may include affinity binding of the fragment from the lysis mixture.

[0009] In another aspect, the present disclosure provides a recombinant antigen comprising the peptide with SEQ ID NO. 1 or at least 80% amino acid sequence identity to the peptide with SEQ ID NO. 1 , wherein the antigen is not glycosylated. In some preferred embodiments, the antigen may be bound to solid support.

[0010] In yet another aspect, the present disclosure provides a method for detecting SARS-CoV-2 antibodies in a biological sample, the method comprising contacting the antigen obtaining by the method of claim 1 with a biological sample and detecting antigen/antibody complexes. In some preferred embodiments of the method, the antigen may be bound to a solid support and the detection of the complexes comprises incubating with a secondary antibody linked to an enzyme, providing a substrate for the enzyme and measuring the substrate degradation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1. Expression, purification, and refolding of S319-640. a) Expression of S319-640 (arrow) in BL21 (DE3) in the absence of IPTG (lane 1 ) or in the presence of 0.2 mM IPTG for one hour (lane 2) or two hours (lane 3) with no protein observed after extraction in 6 M urea in the absence of BugBuster reagent (lane 4). b) S319-640 (arrow) extracted in 6 M guanidium chloride diluted in 6 urea (lane 1 ) subsequently applied to a Na2+-NTA column. Protein was primarily contained in the flow-through (lane 2) with none observed in the column wash in 6 M urea (lane 3) or elution with 0.2 M imidazole (lane 4). c) Solubilization of S319-640 (arrow) with BugBuster reagent in combination with successive urea washes and sonication with the whole cell lysate supernatant (lane 1 ) and 2 M urea wash supernatant (lane 2) lacking in protein but observation of successful extraction in the 6 M urea wash supernatant (lane 3). d) On-column refolding of S316-640 (arrow) first diluted to 2 M urea (lane 1 ) and applied to a His-Trap affinity purification column.

[0012] Figure 2. Antigenic properties of the refolded surface glycoprotein fragment. The antibody binding by the refolded 319-640 SARS-CoV-2 surface glycoprotein fragment (bRBD (1 ) and bRBD (2)) was compared to antibody binding by the 319-591 fragment expressed in human cells (hRBD).

DETAILED DESCRIPTION

[0013] SARS-CoV-2 is an enveloped single-stranded RNA virus (Lu et al. 2020). An RNA-based metagenomic next-generation sequencing approach has been applied to characterize its entire genome, which is 29,881 bp in length (GenBank no. MN908947), encoding 9860 amino acids (Chen et al. 2020). SARS-CoV-2 encodes 4 structural proteins: the surface glycoprotein S which can be also referred to as spike, the envelope protein E, the membrane protein M and the nucleocapsid protein N. (Chan et al. 2020).

[0014] In one aspect, this disclosure provides methods for producing an antigen, wherein the antigen may comprise one or more of: the surface glycoprotein S or any fragment thereof, the envelope protein E or any fragment thereof, the membrane protein M or any fragment thereof, or the nucleocapsid protein N or any fragment thereof in bacterial cells. In some embodiments, the methods include transformation of bacterial cells, preferably E.coli, with a recombinant construct, preferably a plasmid comprising a promoter, preferably an inducible promoter. The promoter controls expression from a DNA sequence encoding one or more of the S protein or any fragment thereof, the E protein or any fragment thereof, the M protein or any fragment thereof, or the S protein or any fragment thereof. Preferably, the DNA sequence is fused in frame with one or more purification tags, preferably a poly-histidine purification tag. [0015] In some embodiments, the methods further comprise culturing the transferred cells and lysing the cells with one or more cell lysis buffer and by sonication. In some preferred embodiments, the one or more proteins or any fragment thereof are produced in inclusion bodies. Preferably, the transfected cells containing the inclusion bodies are contacted with one or more lysis regents which may comprise one or more detergents and/or enzymes for rapturing bacterial walls and then sonicated. This extraction procedure may be repeated several times. The extraction procedure produces a cell lysate mixture comprising the one or more of the proteins and/or protein fragments. The proteins and/or the protein fragments are then purified from the lysate mixture. Various purification methods can be used and preferably include purification by affinity binding of the protein or its fragment. In some preferred embodiments, purification includes contacting the lysate mixture with a resin comprising Nickel ions to which the protein or its fragment can be bound if the protein or its fragment contains a poly-histidine tag. In some embodiments, purification can be conducted simultaneously with refolding the protein or its fragment. The refolding can be conducted by diluting with a refolding buffer, which preferably contains reduced glutathione, oxidized glutathione (GSSG) and urea, applying the diluted lysate to an affinity column and binding the protein or its fragment to the column, and then by gradually decreasing the concentration of urea through several consecutive washes.

[0016] In some preferred embodiments the present methods may be used for producing an antigen which contains at least a fragment of the SARS-CoV-2 surface glycoprotein.

[0017] It has been reported that the total length of SARS-CoV-2 surface glycoprotein is 1273 aa and consists of a signal peptide (amino acids 1—13) located at the N-terminus, the S1 subunit (14-685 amino acid residues), and the S2 subunit (686-1273 amino acid residues); the last two regions are responsible for receptor binding and membrane fusion, respectively. In the S1 subunit, there is an N-terminal domain (14-305 amino acid residues) and a receptor-binding domain (RBD, 319-541 amino acid residues); the fusion peptide (FP) (788-806 amino acid residues), heptapeptide repeat sequence 1 (HR1 ) (912-984 amino acid residues), HR2 (1163- 1213 amino acid residues), TM domain (1213-1237 amino acid residues), and cytoplasm domain (1237-1273 amino acid residues) comprise the S2 subunit. (Huang, Y., 2020). [0018] In one embodiment, the present disclosure provides a method for producing in bacterial cells a SARS-CoV-2 surface glycoprotein fragment which comprises at least a portion of the S1 subunit, and preferably at least a portion of a receptor-binding domain (RBD) and at least 20 amino acid residues located C- terminally from the RBD domain, for example at least amino acid residues 542 through 562, 542 through 572, 542 through 572, 542 through 582, 542 through 592, or 542 through 640. Unlike the naturally occurring SARS-CoV-2 surface glycoprotein (spike) which is known to be glycosylated, the recombinant SARS-CoV-2 surface glycoprotein fragments of this disclosure which are produced in bacterial cells are not glycosylated. [0019] The preferred recombinant SARS-CoV-2 surface glycoprotein fragments of this disclosure comprise at least one purification tag for purification by affinity binding. Preferably, the purification tag is a poly-histidine tag. The methods include growing bacterial cells which express one or more of the SARS-CoV-2 surface glycoprotein fragments, preferably inducing expression of the one or more fragments from the inducible promoter, harvesting the cells, contacting the cells with a lysis buffer and sonicating the cells. The lysis buffer may contain one or more of the following: a detergent, a salt, and/or one or more enzymes, e.g. lysozyme, for rapturing the bacterial walls. In preferred embodiments, BAGBUSTER™ Protein Extraction Reagent (Millipore Sigma) can be used as a lysis buffer.

[0020] The methods may further comprise purifying the fragment by affinity binding and refolding the fragment. Various purification methods can be used and preferably include purification by affinity binding of the protein or its fragment. In some preferred embodiments, purification includes contacting the lysate mixture with a resin comprising Nickel ions to which the protein or its fragment can be bound if the protein or its fragment contains the poly-histidine tag. In some embodiments, purification can be conducted simultaneously with refolding the protein or its fragment. The refolding can be conducted by diluting with a refolding buffer, which preferably contains reduced glutathione, oxidized glutathione (GSSG) and urea, applying the diluted lysate to an affinity column and binding the protein or its fragment to the column, and then by gradually decreasing the concentration of urea through several consecutive washes.

[0021] One preferred antigen according to this disclosure is shown below as SEQ ID NO. 1. It comprises amino acid residues 319 through 640 of the SARS-CoV-2 surface glycoprotein and a histidine purification tag which can be replaced with any other suitable purification tag.

SEQ ID NO. 1

[0022] The SARS-CoV-2 surface glycoprotein fragment with the SEQ ID NO. 1 contains the RBD domain (amino acid resides 319-541 ) and amino acid residues 542 through 640 of the SARS-CoV-2 surface glycoprotein.

[0023] The proteins or protein fragments produced according to the methods of this disclosure are suitable as reagents for testing bodily fluids, preferably blood serum or saliva, for presence of an antibody specific for SARS-CoV-2 virus. Accordingly, the present disclosure provides methods, including serological testing methods. In some embodiments, these methods may comprise contacting one or more antigens of this disclosure, and preferably, the antigen with SEQ ID NO. 1 with a biological sample, preferably blood serum, and detecting the antigen/antibody complexes if the biological sample contains SARS-CoV-2 antibody. The antigen may be bound to a solid support. The antigen/antibody complexes may be detected by any method, including in an Enzyme-Linked Immunosorbent Assay (ELISA). Other detection methods can be also used.

[0024] In another aspect, the present disclosure provides a comparative analysis of antigenic properties between two surface glycoprotein fragments. The first antigen is a fragment comprising amino acid residues 319-591 of the SARS-CoV-2 surface glycoprotein and the second antigen comprises amino acid residues 319-640 of the SARS-CoV-2 surface glycoprotein. The former is commercially available from GenScript and was successfully expressed in HEK293 cells and purified from the conditioned media of the transiently transfected cells. The latter could not be detected in the conditioned media of HEK293 cells transiently transfected with the corresponding expression constructs based on two different vectors. Therefore, it was expressed in E. coli cells.

[0025] The SARS-CoV-2 surface glycoprotein fragment (S319-640) was expressed at high levels in BL21 (DE3) cells (Figure 1a) but was found to be primarily contained within inclusion bodies as indicated by the firm white pellets obtained after cell lysis Extraction of the protein from inclusion bodies was not effective with urea alone (Figure 1a), but was effective with high concentrations (6 M) of guanidium chloride (Figure 1b). Despite this effective solubilization, the protein could not be recovered upon removal of guanidium chloride, even with intermediate concentrations of urea to aid in refolding (Figure 1b).

[0026] BUGBUSTER™ is a detergent based solubilization reagent that has been shown to be effective in extraction of proteins from inclusion bodies (1 ). Incubation with BUGBUSTER™, in combination with sonication steps in the presence of increasing concentrations of urea allowed for extraction of S319-640 from E. coli (Figure 1c). The protein extracted from this procedure was stable for several weeks at 4 in 6 M urea, but could not be diluted below 2 M urea without an optimized refolding procedure.

[0027] The protocol for the refolding of S319-640 was adapted from Zhao et al. which utilized gradual decreases in urea concentrations in combination with oxidized and reduced glutathione to allow for proper formation of disulfide bonds along with glucose and glycerol as stabilizing agents to refold a fragment of the SARS-CoV-1 surface glycoprotein (2).

[0028] We devised a Ni²⁺-NTA column-based refolding procedure to allow for simultaneous purification and refolding of the S319-640 protein. In this procedure protein was applied to the column in the presence of 2 M urea, after which the urea concentration was stepped down in 0.5 M increments with slow flow rates (0.2-0.6 mL/min) until there was no remaining urea.

[0029] A significant amount (>50%) of the protein was contained in the flow- through (Fig. 1 d, lane 2), likely due to the presence of contaminating inclusion bodies that were not fully solubilized. The column was washed with 30 mM imidazole (lane 3) following the refolding procedure and eluted with 0.5 CV fractions of 0.5 M imidazole in refolding buffer (lanes 4, 5 and 6 of Figure 1d). About 10% of the loaded protein was eluted from the column in non-denaturing conditions. Protein obtained from refolding procedure was stable at 4

for several weeks and was used for detection of SARS-CoV-2 antibodies in human blood serum samples (Figure 2).

[0030] The refolded recombinant surface glycoprotein fragment S319-640 recognized the antibodies specific to SARS CoV-2 in a manner similar, but not identical, to S319-591 fragment expressed in human cells. Figure 2 summarizes results of two assays, in which three sets of human blood serum samples were analyzed.

[0031] Samples 13579 and 7489 serve as negative controls. These serum samples were collected from asymptomatic individuals, and never showed any reactivity to either SARS-CoV-2 nucleocapsid protein or to S319-591 surface glycoprotein fragment.

[0032] Samples 2109 and 2104 were collected from a male and a female, a couple who recovered from COVID-19. For both, the diagnosis was confirmed by a PCR test. Both have a high amount of antibodies binding to surface glycoprotein fragments expressed in human or bacterial cells (Figure 2, compare the blue, orange, and grey bars).

[0033] Their asymptomatic young children, a male (2107) and a female (2101 ), who are members of the same household, were SARS-CoV-2 negative according to a PCR test performed at the same time as their parents' PCR test. Nevertheless, 2107 has antibodies specific to S319-591 expressed in human cells and to S319-640 expressed in E. coli. 2101 serum does not have any reactivity to S319-591 expressed in HEK293 cells, but clearly shows reactivity to S319-640 expressed in E. coli. It remains to be established whether this observed difference is caused by an epitope formed by amino acid residues in the 592-640 region, by absence of glycosylation of the protein fragment expressed in bacteria, or by combination of both factors.

[0034] In one preferred embodiment, this disclosure provides a procedure for purification of SARS-CoV-2 surface glycoprotein fragment containing amino acid residues 319-640 from inclusion bodies in E. coli. Although the protein yield is low (5- 10%), the recombinant fragment serves as a potent antigen for serological tests that detect SARS-CoV-2 antibodies in a subject’s sample.

Example 1. Antigen Expression in E. coli cells and Protein Extraction

[0035] BL21 (DE3) E. coli cells were transformed with a pET30a+ expression vector encoding residues 319-640 of SARS-CoV-2 surface glycoprotein (SEQ ID NO. 1 ). Cells were grown to an OD₆₀₀ of 0.5 and induced with 0.2 mM IPTG at 37 ºC.

[0036] After two hours the cells from 1 L of culture were harvested at 10,000 rpm in a Beckman JA-10 rotor for 10 minutes and solubilized with 8 mL BUGBUSTER™ Protein Extraction Reagent (Millipore Sigma) per liter of culture for 30 minutes at 30 °C. 15 mL Base Buffer (20 mM Tris pH 8, 0.5 M NaCI, 10% glycerol and 5 mM [3- mercaptoethanol) per liter of culture was added to the solubilized cells and sonicated on ice 15 x 20 seconds with a 50% duty cycle at 75% power. The resulting suspension was centrifuged for 10 min at 10,000 rpm at 4° C. The pellet was resuspended in 15 ml per liter of culture in Base Buffer supplemented with 2 M urea and homogenized with a glass dounce homogenizer. The sonication and centrifugation procedures were repeated, and the resulting pellet was resuspended by dounce in 15 mL per liter of culture of Base Buffer supplemented with 6 M urea. The pellet was again subjected to the same sonication and centrifugation procedures and the supernatant was collected.

Example 2. Antigen Refolding and Purification

[0037] The extracted supernatant was diluted 10-fold in Refolding Buffer (20 mM Tris pH 8, 20% glycerol, 55 mM glucose, 0.5 M NaCI, 2 mM reduced glutathione (GSH) and 0.2 mM oxidized glutathione (GSSG)) supplemented with 2 M urea. The diluted extract was applied to a HisTrap HP column (Millipore Sigma) at 1 mL/min at room temperature. The column was washed with 2 CV of Refolding Buffer supplemented with 1 .5 M urea at 0.2 mL/min for the first 0.5 CV and 0.6 mL/min for the remaining 1 .5 CV. This procedure was repeated with Refolding Buffer supplemented with 1 M urea, 0.5 M urea and 0 M urea. Following the refolding procedure, the column was washed with Refolding Buffer supplemented with 30 mM imidazole and eluted with the same buffer supplemented with 0.5 M imidazole.

Example 3. Enzyme-Linked Immunosorbent Assays with Recombinant Antigen

[0038] The 96-well Immulon 4 HBX plates (ThermoFisher Scientific #3855) were coated with 0.1 pg of antigen produced according to Examples 1 and 2 (the antigen with SEQ ID NO. 1 ), and dissolved in phosphate buffered saline (PBS), pH 7.4 per well overnight. The plates were blocked with 3% non-fat milk in PBS, pH 7.4, containing 0.1 % Tween-20 (PBS-T), washed with PBS-T, and incubated with serum samples diluted 1 :50 in PBS-T containing 1 % dry milk. The bound IgG antibodies were detected using horseradish peroxidase (HRP)-conjugated monoclonal antibodies recognizing an Fc domain of human IgG (GenScript, # A01854).

[0039] The blood serum samples were collected from donors recovered from COVID-19 (the diagnosis confirmed by an RT-PCR test) and their family members. The blood draws were performed after the symptoms of the disease subsided and over 4 weeks after the disease onset.

References

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Claims

Claims What is claimed is:

1 . A method for producing an antigen for serological tests that detect SARS-CoV- 2 antibodies, the method comprising: a) growing bacterial cells which express a recombinant SARS-CoV-2 surface glycoprotein fragment fused to a purification tag, b) inducing expression of the fragment, c) harvesting the cells, d) resuspending the cells in a lysis buffer and sonicating this lysis mixture; and e) purifying and refolding the antigen from the lysis mixture.

2. The method of claim 1 , wherein the fragment is a peptide with SEQ ID NO. 1 or a peptide with at least 80% amino acid sequence identity to the peptide with SEQ ID NO. 1.

3. The method of claim 1 , wherein purification includes affinity binding of the fragment from the lysis mixture.

4. A recombinant antigen comprising the peptide with SEQ ID NO. 1 or at least 80% amino acid sequence identity to the peptide with SEQ ID NO. 1 , wherein the antigen is not glycosylated.

5. The recombinant antigen of claim 4, wherein the antigen is bound to solid support.

6. A method for detecting SARS-CoV-2 antibodies in a biological sample, the method comprising contacting the antigen obtaining by the method of claim 1 with a biological sample and detecting antigen/antibody complexes.

7. The method of claim 6, wherein the antigen is bound to a solid support and the detection of the complexes comprises incubating with a secondary antibody linked to an enzyme, providing a substrate for the enzyme and measuring the substrate degradation.