Epitope-based Vaccine Design from Alpha and Beta Variant of

SARS-CoV-2: An Immunoinformatics Approach

Hendyco Pratama¹, Nur Imaniati Sumantri¹*, Siti Fauziyah Rahman¹, Viol Dhea Kharisma^2,3,

Arif Nur Muhammad Ansori^4,5,6

¹Department of Electrical Engineering, Faculty of Engineering, Universitas Indonesia, Depok, Indonesia.

²Computational Virology Research Unit, Division of Molecular Biology and Genetics,

Generasi Biologi Indonesia Foundation, Gresik, Indonesia.

³Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia.

⁴Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia.

⁵European Virus Bioinformatics Center, Jena, Germany.

⁶Uttaranchal Institute of Pharmaceutical Sciences, Uttaranchal University, Dehradun, India.

ABSTRACT:

Coronavirus disease 2019, also known as COVID-19, is a respiratory disease. Symptoms of COVID-19 include fever, dry cough, inflammation of the throat area, loss of smell, and even breathing difficulty. COVID-19 is caused by SARS-CoV-2 infection, a virus that is a member of the coronavirus family. The SARS-CoV-2 structure consists of S (spike), M (membrane), E (envelope), and N (nucleocapsid) protein. Two SARS-CoV-2 variants, namely alpha (B.1.1.7) and beta (B.1.351) variants are considered a variant of concern (VoC) due to their increased infectivity. It has been reported that the vaccine's efficacy against these two variants decreased. The purpose of this study is to compare epitopes from S and N proteins of alpha and beta variants to find the most suitable vaccine candidate through reverse vaccinology. In this study, physicochemical properties, antigenicity, and epitope prediction, as well as molecular docking of the epitope and B cell receptor, 5IFH, were done. The result suggested that the epitope from S protein was more suitable as a vaccine candidate. S protein epitope has a lower global energy value which means that it can bind to 5IFH more spontaneously compared to N protein epitopes. The most suitable vaccine candidate for the alpha variant is Pep_B, with a global energy value of -48.77 kcal/mol, and Pep_F, for the beta variant, with a global energy value of -61.61 kcal/mol. These results would recommend the epitopes to be used in further COVID-19 vaccine development. 

INTRODUCTION: 

Coronavirus disease 2019, also known as COVID-19, is a human respiratory disease caused by SARS-CoV-2 infection¹. SARS-CoV-2 was first discovered in Wuhan, China, in December 2019^2,3. The first case of COVID-19 outside of China was reported on 13 January 2020 in Thailand. As of now, COVID-19 has spread globally and reached a total of more than 4 million reported cases, with more than one hundred thousand deaths.

SARS-CoV-2 is a species of coronavirus which belongs to the genus betacoronavirus^4,5. Multiple studies have reported that this virus has a similarity with SARS-CoV, the virus responsible for the severe acute respiratory syndrome (SARS) in China, 2002, and MERS-CoV, the virus responsible for Middle East Respiratory Syndrome (MERS) back in 2012 at Middle East countries⁶. SARS-CoV-2 also has a similar structure as SARS-CoV and MERS-CoV. It consists of four main structures, the spike or S protein, the nucleocapsid or N protein, the envelope or E protein, and the membrane or M protein^5,6.

The S protein is mainly responsible for viral entry. The S1 subunit of the S protein contains the receptor binding site (RBD). The binding of the RBD with angiotensin-converting enzyme 2, ACE2, marks the start of the viral entry⁷. Furthermore, most of the mutations that SARS-CoV-2 underwent occurs in the S protein. Meanwhile, the N protein is considered more stable and conserved compared to the other protein. The amino acid of the N protein has a homology of about 90% and underwent fewer mutations than the S protein⁸.

Multiple variants of SARS-CoV-2 have been reported, and World Health Organization (WHO) categorized said variants into a variant of interest (VoI), a variant of concern (VoC), and a variant of high consequences (VOHC). The most dangerous among these variants are the VoHC, and the least dangerous is VoI, but there has not been any variant considered as VoHC. Meanwhile, two of the variants belonging to VoC are alpha and beta variants⁹. These two variants are characterized by their increased transmissibility, more dangerous symptoms, and their abilities to evade the immune response. Vaccines' efficacy and efficiency have also been reported to decrease up to 30% against these two variants¹⁰.

Studies regarding SARS-CoV-2 vaccine design through in silico method have been conducted multiple times, but most of them use the wild-type isolates of SARS-CoV-2. Enayatkahni et al. conducted a multi-epitope vaccine design derived from SARS-CoV-2 N protein, M (membrane) protein, and ORF3a (open reading frame)¹¹. Jena et al. also conducted a study on a multi-epitope vaccine design derived from SARS-CoV-2 S, M, and N protein¹². Gupta et al. designed a vaccine from SARS-CoV-2 S protein and reported that the vaccine could induce a high cell-mediated immune response^13,. Sarkar et al. designed a multi-epitope vaccine that was reported to be effective against SARS-COV-2 infection from the S, M, and N proteins as well as ORF3a¹⁴. Studies regarding a variant-specific vaccine are very limited.

Due to these reasons, this research was conducted to design an epitope-based vaccine for alpha and beta variants of SARS-CoV-2 through in silico method using an immunoinformatic approach. The vaccine candidates predicted in this study were derived from the S (spike) and N (nucleocapsid) protein of SARS-CoV-2. The S protein was chosen because of its nature of this protein which is known as the very first part of SARS-CoV-2 to bind with angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor in the human body^14,15. Not only that, but most of the mutation also found in these two variants occurs in the S protein¹⁶. On the other hand, N protein was chosen because of its high antigenicity as well as its ability to induce a strong and long-lasting immune response¹⁷.

MATERIALS AND METHODS:

Phylogenetic Analysis:

Phylogenetic analysis for the alpha and beta variants of Indonesian SARS-CoV-2 isolate, EPI ISL 6827364 and EPI ISL 3138807, was conducted using MEGA X software¹⁸. The phylogenetic analysis was done between isolates' proteins with the other 50 isolates acquired from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov) database.

Protein Sequence Acquisition:

The S and N protein of each variant was acquired through NCBI database¹⁹. The accession code for the alpha variant S protein is QWA53375.2, alpha variant N protein is QYC97632.1, beta variant S protein is QWW93436.1, and beta variant N protein is QWW93444.1.

3D Modelling of the Protein:

Models of the proteins were acquired with SWISS-MODEL²⁰. The basic parameter settings are used to model the protein.

Antigenicity and Physicochemical Properties Prediction:

The antigenicity of all proteins was predicted using Vaxijen v2.0 with its default parameter²¹. Physicochemical properties of each protein were predicted using ExPasy's online tool ProtParam²². The physicochemical properties predicted include the number of amino acids, molecular weight, theoretical pI, instability index, aliphatic index, and Grand Average of Hydropathicity (GRAVY).

B Cell Epitope Prediction:

The epitope of each protein was predicted using the IEDB webserver (http://tools.iedb.org/) with the Bepipred linear epitope prediction 2.0 method²³.

Antigenicity, Allergenicity, and Toxicity Prediction of Epitopes:

After acquiring the epitopes of each protein from IEDB, their antigenicity, allergenicity, and toxicity were analyzed. Antigenicity of the epitopes was predicted with Vaxijen v2.0, the allergenicity of the epitopes was predicted with AllerTOP v2.0²⁴, and toxicity with ToxinPred²⁵. The epitopes that will be continued to the next step are the ones that are antigenic, non-allergenic, and non-toxic.

Molecular Docking:

The epitopes selected in the previous step were used for molecular docking. The molecular docking was done between the B cell receptor and the epitopes. First, the epitopes were modeled with PEP-FOLD3^25, and then the B cell receptor (5IFH)^26,27 were acquired from RCSB PDB. The web servers Patchdock^28,29 and Firedock^30,31 were used for the molecular docking process and visualization was performed using PyMol v2.4.0³².

RESULTS AND DISCUSSION:

In this study, the genetic variation of S and N protein of SARS-CoV-2 Alpha and Beta were investigated through phylogenetic analysis. The phylogenetic analysis shows the evolutionary relationship of the isolates depicted with the branches formed. 50 unique isolates from all around the world, including the UK, USA, Italy, Kenya, Philippines, Hongkong, Saudi Arabia, New Zealand, and Germany, are involved in each phylogenetic analysis. As the virus spreads from individual to individual, it undergoes a series of mutations in viral genes that encodes ORF1ab, ORF3a, N, and S proteins. Among these mutations, the mutations that occur in the S protein are considered important since the S protein of SARS-CoV-2 plays a crucial role in the first step of viral mutations³³.

Figure 1 shows the phylogenetic tree for the SARS-CoV-2 Alpha variant for both the S and N protein isolates acquired in Indonesia. The figure compares the similarity between the Indonesian isolates to other sequences gathered from NCBI. As the figure suggests, the S protein of the isolate is close to the isolates gathered from the United Kingdom, United States of America, Italy, and Kenya. But out of all isolates, the S protein of the Indonesian isolate, EPI ISL 6827364, has a striking resemblance with the isolate OU228723.1, which was gathered from England, United Kingdom. On the other hand, the N protein of said isolate has a closer resemblance to isolate OU155876.1, which was gathered from Germany.

Figure 1. Phylogenetic tree of S (left) and N (right) protein of SARS-CoV-2 Alpha variant Indonesian isolates aligned with 50 other isolates.

Figure 2 shows the phylogenetic tree for the SARS-CoV-2 Beta variant for both the S and N protein isolates acquired in Indonesia. As the figure suggests, the S protein of the isolate is close to the isolates gathered from the United Kingdom, United States of America, Germany, Saudi Arabia, Philippines, New Zealand, and Hongkong. But out of all isolates, the S protein of Indonesian isolate, EPI ISL 3138807, has a striking resemblance with the isolate OK104999.1, which was gathered from Texas, United States of America, and the N protein has a closer resemblance to isolate OK006997.1 which was gathered from Georgia, United States of America.

Figure 2. Phylogenetic tree of S (left) and N (right) protein of SARS-CoV-2 Beta variant Indonesian isolates aligned with 50 other isolates.

The sequences gathered from NCBI have their own respective accession code. The accession code for S protein of alpha variant is QWA53375.2, N protein of alpha variant is QYC97632.1, S protein of beta variant is QWW93436.1, and N protein of beta variant is QWW93444.1. These four proteins will be modeled with SWISS-MODEL. Figure 3 shows the model acquired for each protein.

Figure 3. 3D model of (A) S protein of alpha variant, (B) N protein of beta variant, (C) S protein of alpha variant, and (D) N protein of beta variant.

The phylogenetic analysis could show the migratory histories, founder events, and sample size^34,35,36. According to the research, the isolates collected in Indonesia are largely comparable to those collected in the United States of America and the United Kingdom. The SARS-CoV-2 Alpha variant was initially reported in the United Kingdom in November 2020^37,34. The close resemblance of the S protein of EPI ISL 6827364 with OU228723.1 suggests that the S protein of the Indonesian isolates underwent similar mutations to the isolate from the United Kingdom or that EPI ISL 6827364 originated from the United Kingdom. The N protein of said isolates is closer to OU155876.1 from Germany, but nonetheless, the alignment shows that it still closely resembles OD994453.1 from the United Kingdom. Hence, it is still probable that the isolates in Indonesia originated from the United Kingdom.

The phylogenetic analysis for EPI ISL 3138807, Indonesian isolates of SARS-CoV-2 Beta variant, shows that it is mostly similar to the isolates from the United States of America and the United Kingdom. Both the S and N proteins have a strong similarity with the isolates gathered from the United States of America, OK104999.1 and OK006997.1, respectively. On the contrary, the first reported beta variant was from South Africa^37,34. This might be caused by the submitted samples from the United States of America, and the United Kingdom outnumbered the samples from South Africa. The other possibility is that after a series of migrations, the isolates from Indonesia might undergo mutations closer to the beta variant found in the USA rather than South Africa.

The antigenicity and physicochemical properties of all proteins were predicted. Table I shows the results of the predictions.

Table 1 Physicochemical Properties and Antigenicity Prediction of all Protein

QWW93444.1

The value of the alpha and beta variants does not have a big difference in all parameters. An instability index below 40 determines the protein's stability, while more than 40 means it is unstable^35,36. A negative value of GRAVY indicates that the protein is hydrophilic, and a positive value indicates hydrophobicity^36,38. Antigenicity above 0,4 indicates antigenic properties. Based on the prediction, the S protein of both variants is stable, hydrophilic, and antigenic, while the N protein of both variants is unstable, hydrophilic, and antigenic.

From the physicochemical properties, S protein from both variants has 1,270 amino acids, and N protein in both variants has 419 amino acids. The molecular weight of both proteins from both variants also has a similar value. The molecular weight of the S protein of the alpha variant is 140,872.20 Da, and the beta variant is 140,817.12 Da. Meanwhile, the molecular weight of the N protein of the alpha variant is 45,695.92 Da, and the N protein of the beta variant is 45,637.75 Da. The molecular weight is proportional to the number of amino acids present in the proteins and could be predicted from the amino acid inside the proteins³⁸. The mutation caused a slight change in the amino acid inside the proteins, which could cause a small difference in the molecular weight value.

Theoretical pI or isoelectric point shows a pH where the protein has a zero net charge³⁹. When the protein is in a solution with pH below its pI, the protein will have a negative net charge. The same is applied when a protein is in a solution with pH above its pI, and the protein will have a positive net charge. A pI above 7 shows that the protein is basic and below 7 shows that the protein is acidic⁴⁰. The isoelectric point of both proteins from both variants shows that S proteins were acidic and N proteins were basic, with the value of 6.35 for S protein of alpha variant, 6.64 for S protein of beta variant, 10.09 for N protein of alpha variant, and 10.07 for N protein of beta variant. The isoelectric point also indicates that S protein will precipitate in an acidic solution and N protein will precipitate in a basic solution^41,42.

The instability index indicates the stability of a protein. The more stable a protein is, the harder they become denatured. The S protein of both variants is stable, while the N protein of both variants is unstable. The aliphatic index shows the relative volume of proteins occupied by the aliphatic amino acid chain, including alanine, isoleucine, leucine, proline, and valine. The higher the aliphatic index means that the protein is more hydrophobic and thermally stable⁴¹. The S protein of both variants has a higher aliphatic index value compared to the N protein. This indicates that S proteins are more hydrophobic and thermally stable than N proteins.

The grand average of hydropathicity (GRAVY) shows whether a protein is hydrophobic or hydrophilic. It is calculated based on the hydropathy value of the amino acid divided length of the sequence^36,37. All proteins, S protein of both variants and N proteins of both variants, are hydrophilic since they all have a negative value of GRAVY. But the N proteins are more hydrophilic than the S proteins. Antigenicity shows how reactive the epitope is to the antibody molecule. It shows whether the epitope will be recognized as an antigen or not by the immune system⁴³. All proteins, S proteins of both variants, and N proteins of both variants are antigenic, although N proteins are more antigenic than S proteins.

The B Cell epitope of all proteins will be predicted with IEDB. Figure 4 shows the number of epitopes that each protein has. S protein of alpha variant has 35 epitopes, N protein of alpha variants has 11 epitopes, S protein of beta variant has 36 epitopes, and N protein of beta variant has 11 epitopes. The yellow part of the graphs in Figure 4 indicates the part of the protein that is an epitope, while the green part indicates the part of the protein that is not an epitope.

Figure 4. IEDB epitope prediction for (A) S protein of alpha variant, (B) N protein of beta variant, (C) S protein of beta variant, and (D) N protein of beta variant.

The antigenicity, allergenicity, and toxicity of the epitopes acquired from the step before will be predicted for a further selection of the epitopes. Table II shows the remaining epitopes after the selection. After the selection, each protein had only two epitopes left and was named Pep_A to Pep_H.

Table 2: Physicochemical Properties and Antigenicity of Selected Epitopes

Molecular docking was done between Pep_A to Pep_H with a model of an antigen-binding fragment of B cell receptor acquired from RCSB PDB, 5IFH. Fig. 5. shows where each of the epitopes from alpha variant bonds with 5IFH. While Fig. 6. shows the bond of the epitopes from beta variants. Pep_A formed six bonds with 5IFH (SER-153, ILE-154, SER-168, GLN-170, SER-174, and GLN-207), and Pep_B formed seven bonds (with LYS-105, THR-111, THR-166, PRO-167, LYS-169, SER-174, and TYR-181), Pep_C formed two bonds (VAL-149 and THR-164), Pep_E formed three bonds (ASP-152, SER-153, and THR-166), Pep_F formed eight bonds (GLY-40, ASP-84, THR-111, ASP-152, SER-153, GLN-170, PHE-172, and SER-174), Pep_G formed four bonds (SER-25, GLN-41, GLN-108, and SER-171), and Pep_D, as well as Pep_H, formed no bond at all.

Figure 5. A bond formed between (A) Pep_A, (B) Pep_B, (C) Pep_C, and (D) Pep_D with 5IFH.

Figure 6: A bond formed between (A) Pep_E, (B) Pep_F, (C) Pep_G, and (D) Pep_H with 5IFH.

The global energy of each pair is calculated using the firedock webserver. Table III shows the global energy of each epitope. Global energy value shows Gibbs free energy, where it is preferable to have a low value^42,43,44. Negative Gibbs free energy, which indicates that a process is exogenic and does not require external energy to occur, is in accordance with the thermodynamic law. In other words, negative Gibbs free energy indicates that a reaction could happen spontaneously^{45,46,47,48,49,50,51,52}. The molecular docking result shows that the epitope acquired the lowest global energy from the S proteins, namely Pep_B with -48.77 kcal/mol for the alpha variant and Pep_F with -61.61 kcal/mol for the beta variant. Overall, the global energy from the beta variant has lower global energy. This means that the beta variant is more likely to bind with the B cell receptor.

Table 3 Global energy value of all epitopes.

Based on the physicochemical properties, both S and N proteins from both variants are hydrophilic and antigenic. But N protein is more hydrophilic and antigenic compared to S proteins. Since N proteins are more hydrophilic, it means that N proteins are more soluble than S proteins. Studies have shown that a soluble antigen has an advantage in inducing immune response^45,46,47. A higher number of immune complexes (ICs) as well as IgG have been reported to be formed in response to soluble antigens. Not only that, but N proteins also have a higher antigenicity than S proteins which mean N protein could induce immune response easily compared to S proteins. Hence, from the physicochemical properties, N proteins are more suitable to be made as a vaccine candidate.

Some studies have shown that the N protein of SARS-CoV-2 is a vaccine candidate with a lot of potentials. N protein has highly immunogenic traits, conserved sequence, as well as expressed a lot in the infection period^47,48. A large number of N protein-specific IgG has also been reported in SARS-CoV-2 patients^48,49. A study on multi-epitope vaccine design with SARS-COV-2 N protein shows that the vaccine developed could elicit humoral and cell-mediated immune response^{49,53,54,55,56}.

Although the physicochemical properties indicate that the N protein is a better vaccine candidate due to its more antigenic and hydrophilic traits, the difference between S and N proteins is insignificant. Furthermore, the molecular docking results show that the epitopes from S protein interact more with B cell receptors than those from N protein. The global energy of the S protein epitopes is also lower than the N protein epitopes. This indicates that S protein epitopes could better bind with B cell receptors than N protein epitopes. Hence B cell will also be activated, and immunity will be formed faster.

A study reported that most vaccines use part of the SARS-CoV-2 S proteins. Pfizer and Moderna use mRNA of SARS-CoV-2 S protein, and AstraZeneca uses a whole SARS-CoV-2 S protein with adenovirus vector ChAdOx1^50,51. It is also reported in previous studies on SARS-CoV that N protein as a vaccine target could not provide immunity and even caused a higher pneumonia rate.

CONCLUSION:

All in all, the research conducted shows that alpha and beta variant does not have that much of a difference in terms of their physicochemical properties, but molecular docking result shows that the beta variant could bind better with B cell receptor. N protein, although not much, is more antigenic and hydrophilic than S protein in both variants. Despite that, the S protein epitope could bind better with B cell receptors than N protein epitopes, indicated by the difference in global energy value where S protein epitopes have lower global energy. Hence, the most suitable candidate for vaccine design is Pep_B (LTPGDSSSGWTA) for the alpha variant and Pep_D (LPDPSKPSKRS) for the beta variant.

ACKNOWLEDGEMENT:

We gratefully acknowledge the funding from Kementerian Pendidikan, Kebudayaan, Riset, dan Teknologi, Republic of Indonesia through Penelitian Dasar Unggulan Perguruan Tinggi 2022 No. NKB-846/UN2.RST/HKP.05.00/2022.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Accepted on 21.02.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(10):4617-4625.