Development of superior antibodies against the S-protein of SARS-Cov-2 using macrocyclic epitopes

2.1 Design of peptides

Conformational searches were performed using the Low-Mode MD application from the “Molecular Operating Environment” software MOE 2016.08 (Computing & Inc, 2016). The reported crystal structure of the SARS-Cov-2 RBD in complex with ACE2 has allowed us to select the sequences with good interactions and can be used as epitopes for neutralizing antibody development (Lan et al., 2020). The topology of these regions was an important parameter for peptide selection. It is worth noting that the used SARS-Cov-2 RBD was the one discovered at the time we started the project, and this is the reason why it was selected in this work. The details are the following: Gene: (S) UNQ 868/PRO1885; Mutations = 0; Uniport code = P0DTC2; PDB ID = 2M0J. The stability of the selected peptides after initial energy minimization and after MD simulation was important parameter as well. In addition, the presence of cysteine residues was another parameter that allowed the formation of macrocyclic peptides through disulfide formation.

2.2 Molecular dynamics

All molecular dynamic (MD) simulations were conducted by MOE software (Computing & Inc, 2016). The dynamics simulations were applied for a conformational search to find out the stable conformation and calculate the RMSD for each designed peptide. The quality of the temperature-related factors, protein geometries, and electron density were tested. All hydrogens were added, and energy minimization was computed. The solvent molecules in the system were deleted before solvation, and salt atoms were added to ensure complete neutralization of the biomolecular system. The solvent atoms were added to surround the system in a cubic shape. Amber 10:EHT was selected as a force field in the potential setup step. All Van der Waals forces, electrostatics, and restraints were enabled. The heat was adjusted to increase the temperature of the system from 0 to 300 K, followed by equilibration. Cooling was then initiated until 0 K was reached. The simulation was conducted over 50 ns period of time. The 3D structure of peptides was built, and the protonation state was checked. Partial charges were calculated. Energy was minimized using a suitable forcefield. The topology problems were solved. The system was surrounded by a cube shape of solvent (water), and NaCl was used as a salt to neutralize charged system. Amber12: EHT was selected as an all-atom forcefield. All bonds, Van Der Waals, electrostatics, and restraints were enabled. Energy minimization was performed. Root mean square deviation gradient was 1.0. Start time was zero, and the checkpoint was 250. The molecular dynamics protocol was done using NPA (Nose-Poincare-Andersen) to solve the equation of motion. Temperature was 300 K, and the time scale was 50000 ps (50 ns).

2.3 Peptide synthesis

One Linear and two macrocyclic counterpart peptides have been synthesized. The synthesis was performed on Rink Amide MBHA Resin using the Fmoc solid-phase peptide approach and orthogonal protecting groups were carefully chosen. The sequences of peptides are presented in Table 1.

Cyclization of peptides was performed through disulfide formation between the side chains of the cysteine amino acids. For example, for the synthesis of the semi cyclic peptide (2), the sequence CNGVEGFNCY-NH₂ was first assembled on resin using orthogonal protecting groups. The oxidization was made then between the two Cysteine on the resin by using Iodine in DMF, and the synthesis was continued after that to complete the desired sequence. The same strategy was applied for the synthesis of the second cyclic peptide (3). The three peptides were cleaved from the Rink amid resin and the protecting groups were removed simultaneously using the mixture TFA/TIS/water (95:2.5:2.5). The three peptides bear a terminal cysteine to conjugate them to the immunogenic BSA protein.

2.4 HPLC analysis

In order to verify the purity of the synthesized peptides, HPLC Analyses were carried out. All the experiments were conducted on CXTH LC3050N system coupled with a UV–Visible detector. Successful purifications of samples were achieved by gradient elution on a reverse-phase Gemini-NX 5μ C- 18 110A, 4.6*250 mm column, using a mobile phase consisting of 0.1 %Trifluoroacetic in 100% Acetonitrile and 0.1 %Trifluoroacetic in 100% Water at a detection wavelength of 220 nm, Flow rate: 1.0 ml/min and volume: 20 µl. CXTH data system software was used to control the HPLC system and for data acquisition and analysis. The gradient mobile phase set for the different peptides is presented in (SI, Table S1). All the peptides were obtained with a purity greater than 90% as shown in the HPLC spectra (SI, Figures S1, S2 and S3).

2.5 LC-MS spectrometry

Peptides’ masses were verified by LC-MS (SI, Figures S4, S5, and S6). The analyses were carried out using Agilent 6125B (ESI) LC/MS. The detection was conducted by an MS equipped with a V electrospray ionization (ESI) source, and the following MS parameters are applied: Nebulizer Gas Flow: 1.5 L/min CDL: −20.0 v CDL Temp: 250 Co Block Temp.: 200 Co Probe Bias: +4.5kv Detector: 1.5 kv and Flow rate: 0.2 ml/min. The separation of samples was achieved by isocratic elution on a reverse-phase Gemini-NX 5μ C- 18 110A, 4.6*250 mm column, using a mobile phase consisting of 50 % H₂O / 50 % acetonitrile. Agilent Open LAB CDS software was used to control the MS data acquisition and analysis.

2.6 Immunization and antibody production

Immunization and boosts (in duplicate) of New Zealand White rabbits for 70 days have been done at Proteogenix (Schiltigheim, France, https://www.proteogenix.science) to produce polyclonal antibodies against the different synthesized epitopes. The experiments on animals were conducting according to the regulations of ISO9001:2015 Quality Management System and the ARRIVE Guidelines (https://arriveguidelines.org). Proteogenix respects the ethical use of animal science and their applied protocols are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Each antibody was produced in duplicate in two different rabbits. For this purpose, six rabbits (randomly female and male) with average body weight 2 kg ± 200 g were used. The rabbits were housed in metal cages separately, where fresh and clean water was supplied. The peptides have been conjugated to a BSA immunogenic protein through the terminal cysteine and injected in the rabbits. The sera have been collected, and biweekly ELISA measurements against the corresponding epitopes were performed to detect the antibody production.

2.7 Direct ELISA on the collected sera

0.5 μg of the RBD was typically immobilized to a 96-well microliter plate via a chemical reaction that results in the covalent attachment of the antigen through free amino groups. Detection is then performed in three steps, which include probing the coated well with equal volumes of sera. This step is followed by probing the bound antibody using a secondary antibody (1:8000) specific for the constant region of the primary antibody. The secondary antibody is typically conjugated to alkaline phosphatase, which can be readily detected using a colorimetric substrate reaction. The color change is then analyzed in a plate reader in a UV–visible spectrophotometer equipped to detect changes in absorbance after different sera dilution. Value of 0.200 in absorbance was considered a threshold of detection.

2.8 Antigen-specific antibody purification

The produced antibodies in the pooled rabbits’ sera were purified using affinity chromatography technique in which antibody-containing sera were filleted to remove any large interfering particles. The sera were then passed through protein G-coupled Sepharose columns then the resulted eluted solutions were passed through agarose beads immobilized with peptides which are specific for the required antibody. Then, the unbounded nonspecific antibodies were washed out with a low salt solution. After that, the targeted antibody is eluted from the beads and assessed for binding to the specific antigen using ELISA. The concentrations of the produced antibodies were determined by UV–visible spectrophotometry and Beer-Lambert equation.

2.9 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

The collected fractions were analyzed by SDS-PAGE under reducing and non-reducing conditions at neutral pH. The concentration of polyacrylamide gel was 12%. Samples were incubated with 2% SDS for 10 mins at 100 °C. Afterward, 200 ng from each sample were loaded onto the polyacrylamide gel wells. The electrophoresis analyses were done using electrophoresis instrument (Bio-Rad Laboratories, USA). After complete separation, the gel was stained with Coomassie Brilliant Blue G 250, and a further distaining step was performed.

2.10 Western blot analysis of antibodies against RBD

The recombinant RBD was purchased from Proteogenix (RBD purity and sequence are presented in SI, Figure S7). RBD was electrophoresed on an 8% SDS-PAGE gel and transferred to a nitrocellulose membrane by blotting. The membrane was blocked by bovine Serum Albumin (BSA) protein. The RBD protein was detected by adding the purified antibodies and washed, then incubated with secondary antibodies and visualized by adding horseradish peroxidase HRP –specific substrate, and the color was developed.

3.1 Peptide design and synthesis

The linear and macrocyclic peptidic sequences have been selected and designed based on the RBD-ACE2 X-Ray structure.(Lan et al., 2020)

The structures of the peptides matching the native sequence in the RBD protein were visualized using MOE software (Computing & Inc, 2016) and are presented in Fig. 1. The synthesis of the designed peptides was successfully achieved with a purity greater than 90%, as calculated from HPLC analyses (SI, Figures S1, S2, and S3). The physicochemical properties of these peptides are presented in SI section (Table S2). The peptides showed isoelectric points less than 7.4 (optimum blood pH), which may correct or antagonize the poor solubility effect.

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Fig. 1

Structures of the designed peptides (1), (2) and (3) matching with the native sequences in RBD.

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3.2 Molecular dynamics

All the designed peptides were subjected to MD simulations over 50 ns period of time. The trajectories were analyzed, and the root mean square deviation (RMSD Å) values were computed. According to the results, the semi-cyclic peptide (2) showed the least RMSD = 0.61 Å compared with 1.52 Å and 2.00 Å for peptides (3) and (1), respectively. Peptide (2) achieved stability after 20 ns (Fig. 2I), and this stability was continuous till the end of MD. Its equilibrium was obtained at least deviation0.61 Å. For peptide (2), it was important to analyze the relaxed and non-relaxed conformations of the cysteine-cysteine bridge before and after equilibrium. The angles were also measured to examine the configuration of the stable conformations (Fig. 2II). The angle of Cys-Cys bridge in (2) was 104.2° and then became 95.0° at equilibrium in its most stable form (Fig. 2II).

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Fig. 2

I) MD of peptide (2) over 50 ns showing the least RMSD = 0.61 Å. II) A) Superimposition of the reference conformation of peptide (2) (red) and the stable one at equilibrium (blue), B) RMSD matrix, C) Measured angle before MD, D) Measured angle after MD. Peptide (1) showed the least stability as it reached equilibrium after a long time of 35 ns (SI, Figure S8). Peptide (3) achieved stability after 30 ns with RMSD 1.52 Å (SI, Figure S9). The angle of Cysteine-Cysteine bridge in (3) was 118.7°, and then it became 102.8°at equilibrium in its most stable form (SI, Figure S9).

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3.3 Polyclonal antibody productions

After immunization of the three synthesized peptides in rabbits for 70 days, all the sera were collected. The ELISA studies against the corresponding epitope were performed to verify the presence of antibodies (SI, Figure S10). The ELISA results confirmed the presence of antibodies in the different collected sera. The extraction of the obtained antibodies has been achieved by purification through antigen conjugated resin which is known to increase the detection sensitivity and reduce the undesirable background. All the antibodies have been purified, and their concentrations have been calculated to be in the range of 1 mg.ml⁻¹. The purified antibodies were obtained in duplicate and denoted A01, A02, and A03 in correspondence with peptides (1), (2), and (3). The antibodies were preserved in PBS buffer, 0.02% sodium azide, pH 7.4. Additionally, their activities against their corresponding epitopes have been verified by ELISA technique. The purity of the different antibodies has been checked by SDS-PAGE in reducing and non-reducing conditions, as shown in Fig. 3.

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Fig. 3

SDS-PAGE for the produced antibodiesA01, A02, and A03 in I) non-reducing and II) reducing conditions. Each antibody has been prepared in duplicate R1 (Rabbit 1) and R2 (Rabbit 2). M is a pre-stained molecular weight marker.

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3.4 Western Blot and ELISA analyses against RBD

To figure out the recognition between the different produced antibodies and the RBD, Western Blot analyses have been performed, as shown in Fig. 4.

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Fig. 4

Western Blot Analyses of antibodies A01, A02, and A03against the RBD protein. Each antibody has been produced in duplicate (Rabbit 1 (R1) and Rabbit 2 (R2)).

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To compare the different antibodies' affinity toward the RBD, ELISA studies have been performed on the three antibodies. The obtained results are shown in Fig. 5.

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Fig. 5

ELISA of the purified antibodies A01, A02 and A03 against the RBD.

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The EC50 or the concentration of antibodies that give half maximal binding were calculated from ELISA curves for A01, A02, and A03. The obtained values were 4.10, 0.39, and 1.30 for antibodies A01, A02, and A03, respectively.