Immunoinformatic design of a COVID-19 subunit vaccine using entire structural immunogenic epitopes of SARS-CoV-2

doi:10.21203/rs.3.rs-27397/v1

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Immunoinformatic design of a COVID-19 subunit vaccine using entire structural immunogenic epitopes of SARS-CoV-2

https://doi.org/10.21203/rs.3.rs-27397/v1

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published 30 Nov, 2020

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Coronavirus disease-19 (COVID-19) is an acute resolved disease, with estimated 3.4% case fatality rate. Due to insufficient data and short onset time of the disease, researchers have to change the strategy against its associated virus, SARS-CoV-2. One of these strategies is the use of computational methods in the field of drug and vaccine design, which can greatly reduce the time and cost of the therapeutic or immunogenic development projects. In this study, we employed various immunoinformatics tools to design a multi-epitope vaccine polypeptide with the highest potential for activating the human immune system against SARS-CoV-2. The initial epitope set was extracted from the whole set of viral structural proteins. Their potential non-toxic and non-allergenic T- cell and B-cell binding and cytokine inducing epitopes were then identified through a priori immunoinformatic prediction. Selected epitopes were bonded to each other with appropriate links. A suitable adjuvant was added to the N-terminus of the vaccine polypeptide sequence to increase its immunogenicity. Molecular modelling of the 3D structure of the vaccine polypeptide, docking, molecular dynamics simulations and free energy calculations confirmed that the designed vaccine had high affinity for Toll-like receptor 3 binding, and that the vaccine-receptor complex was highly stable. Therefore, the designed polypeptide is promising for antigenicity and inducing an effective and safe immune response against SARS-CoV-2 inside the human body.

Pharmacodynamics

SARS-CoV-2

computational methods

immunoinformatics tools

multi-epitope vaccine polypeptide design

Coronaviruses (CoVs) named for the crown-like spikes on their surface are grouped into four main types, known as alpha, beta, gamma and delta1. In recent decades, new human beta-CoVs have evolved from animal-infecting types and led to intercontinental pneumonic outbreaks.

Severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002, and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 engendered epidemics. SARS-CoV-2 was a return of coronaviruses in December 2019 which causes the ongoing coronavirus disease 2019 (COVID-19). The spill-over was from Chinese horseshoe bat SARS-like coronavirus transmitted to humans from a Wuhan seafood market. By 23th April, there have been 2,544,792 cases with 175,694 deaths globally2.

Identifying preventive options, i.e. a COVID-19 vaccine, in a timely manner is critical for the response to the SARS-CoV-2 mass contagion. Live-attenuated vaccines with a long history of success, as well as inactivated whole virus (IWV), DNA, vectored, subunit, and self-assembling virus-like particle vaccines are the diverse candidates for immunogen development, each with particular advantages and disadvantages. Vaccines based on chemically inactivated CoV virions have led to production of neutralizing antibodies with different levels of protection, but they raise potential side effects and biosafety concerns3. DNA vaccines expressing full-length spike protein or its fragments, as well as DNA priming coupled with protein boosting, are also effective against MERS-CoV infection4,5. By intracellular expression of immunogenic antigens, vectored vaccines allow activation of cellular immune response in addition to humoral immunity; they have a proven safety record, however these vectors are limited to presenting one or a reduced number of CoV antigens to the immune system6. Subunit vaccines based on recombinant spike protein has been shown to be very well suited for creating immunogenicity against SARS and MERS7,8. While DNA, vectored and subunit candidates presenting viral epitopes can elicit a focused antibody response, their subviral components may not portray the full antigenic complexity of the virus, resulting in limited protective efficacy or immunopathology due to unbalanced immune responses6. One promising option to confront this issue is the subunit vaccines harbouring numerous and diverse antigenic elements, allowing to produce a more comprehensive spectrum of native viral antigens.

The process of vaccine development takes at least ten years from bench research to approved vaccine use9. Identification of apt targets for developing vaccine immunogens can be promoted using chemistry and topology, and it is becoming more of a rational design exercise which may considerably reduce both the time and costs required for vaccine development and production10. In this study, it was hypothesized that advanced computational screening could identify suitable peptides for the construction of a subunit vaccine. We used the full set of the putative structural proteins from SARS-CoV-2 as targets for creating both B-cell and T-cell immunity. By integrating these peptides together, we develop a multi-epitope-based vaccine polypeptide to bind to Toll-like receptor 3 (TLR-3) and elicit an effective immune response without causing toxicity and sensitivity. We also considered incorporating an adjuvant for augmenting the host antigen-specific immune response. As this method has been validated experimentally with other pathogenic epitopes11, we suggest that the proposed structural formulation would be able to generate an effective immune response against the novel virus, as a vaccine in vivo. The wet lab researchers are expected to validate our design, hoping to reach protection for the healthy community against the COVID-19 pandemic.

The basic steps of the procedure for designing the multi-epitope vaccine are shown in Fig. 1.

Structural T-cell and B-cell epitopes of SARS-CoV-2. The predicted antigenic CTL epitopes harboured in the structural proteins of SARS-CoV-2 are listed in Supplementary Tables S1-S4 online for S, E, M and N proteins, respectively. As well, the IEDB MHC-II prediction tool was applied to predict HTL (15-mer) epitopes and their MHC-II binding. For linear B-cell epitopes the score value > 0.75 was chosen as criteria for selection. Using appropriate server tools, antigenic, non-toxic and non-allergenic T-cell or B-cell epitopes that were able to induce IL-4, IL-10 and IFNγ cytokines were selected for the design of a multi-epitope vaccine. The final selected CTL, HTL, and linear B-cell epitopes are shown in Supplementary Tables S1-S6 online.

Multi-epitope vaccine polypeptide construction. The total of 34 CTL, and 12 HTL epitopic peptides were fused to each other by KK, and GPGPG linkers, respectively, followed by adjoining a single LBL epitope using KK linker, to create the multi-epitope peptide-based vaccine12-14. Furthermore, to boost the immunogenicity of the multi-epitope vaccine, β-defensin (GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK) was added as an adjuvant to the amino terminus of the polypeptide using an EAAAK linker to the first CTL epitope. A total of 694 amino acids were there in the ﬁnal multi-epitope subunit vaccine construct (Fig. 2 and Supplementary Fig. S1 online).

Immunogenicity, allergenicity and physiochemical properties of the vaccine candidate. Assessment of immunogenic, allergenic and solubility indicated a suitable design for the multi-epitope vaccine (Table 1). The calculated molecular weight of the final vaccine (78.35 kDa) indicates its good antigenic nature13,15, and the pI value of 10.38 shows the basic nature of the final vaccine candidate. The instability index of the final polypeptide (36.73) shows its high stability. The GRAVY value was −0.261, showing that the final structure is slightly hydrophilic in nature and can lead to better connection with other proteins. Moreover, the aliphatic index of the peptide is equal to 81.44, which implies the final peptide has a high thermo-stability (Table 1). Estimated half-life was 30 h in mammalian reticulocytes in vitro, and > 20 h in yeast, and > 10 h in E. coli in vivo.

Table 1. Antigenic, allergenic and physiochemical assessments of the primary sequence of final vaccine protein

Features	Assessment
Number of amino acids	694
Molecular weight	78351.68Dalton
Theoretical pI	10.37
No. of negatively charged residues (Asp + Glu)	25
No. of positively charged residues (Arg + Lys)	137
Extinction coefficient (at 280 nm in H2O)	120305 M-1cm-1
Instability index	36.73
Aliphatic index	81.44
Grand average of hydropathicity (GRAVY)	-0.261
Antigenicity	0.7342 (VaxiJen v.2.0)
Allergenicity	Probable non-allergen (AllergenFP v.1.0) Probable non-allergen (AllerTOP v.2.0)
Solubility	0.914870 (SOLpro)

3D structure of the vaccine polypeptide. The GalaxyWEB server was employed for modelling and refinement of the 3D structure of the multi-epitope vaccine polypeptide. The modelled 3D structures were then validated to gain a best model. ProSA-web tools showed a Z- score of -4.33, indicating the good quality of the final vaccine structure (Supplementary Fig. S1 online). In addition, for the best model (Supplementary Fig. S1 online), RAMPAGE analysis indicated that 92.3% amino acids of the final structure were in the favoured area, 6.1% were in the allowed area, and 1.6% were in the disallowed area of Ramachandran plot, reflecting a high structural quality of the constructed vaccine model (Supplementary Fig. S1 online).

Conformational and linear B-cell epitopes in the vaccine polypeptide structure. The total of 7 conformational epitopes with a score of 0.702 to 0.882, and 9 linear epitopes with a score of 0.7 to 0.878 were selected as the final B-cell epitopes (Fig. 3, Supplementary Table S7 online). PI value (the score given by ElliPro) of 0.882 shows that 88.2% residues are locating in the predicted ellipsoid area of the epitope and this epitope features the highest solvent accessibility.

Binding of the vaccine structure to TLR-3. The best docked complex of the immune receptor TLR-3 with the vaccine model was identified from among various server outputs by comparing the binding free energy. Analysis of the residues contributing at the protein-protein interface showed that the C-terminal domain of the polypeptide is involved in the interaction, where residues from vaccine polypeptide form polar or non-polar contacts with three domains on the TLR-3 structure (Supplementary Fig. S2 online). The docked complex was further applied for running MD simulation investigations.

MD relaxation and analysis of the receptor-vaccine complex. The MD simulations of the docked multi-epitope-based subunit vaccine with TLR-3 as the receptor was done to achieve information about the conformational changes of TLR-3-vaccine polypeptide complex. Such studies were essential for several vital facets: (1) to comprehend whether the designed vaccine is stable at the bound pocket; (2) to validate that the induced conformational mobility of both TLR- 3 receptor and the multi-epitope vaccine has not negative impact on the conformation of the docked proteins; and (3) to corroborate that the epitopes of the multi-epitope vaccine are subject to efficient recognition by the human immune system, causing strong immune response.

Three statistical factors were assessed based on 24000 ps of simulation trajectory (Fig. 4). The root mean squared deviation (RMSD) values of TLR-3 and multi-epitope vaccine in the complex reﬂect the high conformational stability of the docked molecules. An average RMSD of 0.29 nm with maximum of 0.44 nm realized at 14000 ps was noted for the TLR-3 molecule (Fig. 4A). The RMSD value for multi-epitope vaccine (Fig. 4B) showed that it mostly remained stable during simulation time, with a plateau at about 10500 ps. The observed trends can be attributed to the moving multi-epitope vaccine at TLR-3 binding pocket in an effort to obtain a suitable and stable docked conformation.

Region-wise structural fluctuations of the TLR-3-vaccine polypeptide complex were studied by calculating the root mean squared fluctuation (RMSF) parameter (Fig. 4C-D). The mean RMSF calculated for TLR-3 and the multi-epitope vaccine are 0.2 nm and 0.83 nm, respectively, which are overall in favour of protein residues local stability. Along the vaccine sequence, residues located at the loop regions (such as I137, T138, W204, N376 and R654) have high fluctuation (Fig. 4D). The flexibility of the loop regions is essential for proper holding the vaccine at the binding pocket.

The compactness of the complex structure was estimated by calculating the radius of gyration (Rg) of TLR-3 and multi-epitope peptide molecules (Fig. 4E-F). The graph shows that during the simulation time, TLR-3 and multi-epitope vaccine molecules have mean Rg values of about 3.56 nm and 4.87 nm, respectively. Rg fluctuations is an indication of movements and conformational changes in flexible regions of the multi-epitope vaccine peptide in the TLR-3 binding pocket. This dynamics seems essential to suitably identify the vaccine and incorporating it in the binding pocket.

Free energy of the binding between vaccine polypeptide and TLR-3. To figure out the strength of the contact between multi-epitope vaccine and TLR-3 structures, the binding free energy between the two molecules was calculated using MMPBSA approach. According to Table 2, the nonpolar element (-136.92 kcal/mol) was an important energy term in the binding free energy of the complex. Our findings clarified that the favourable electrostatic energy (Eele = -241.5 kcal/mol) was covered up by the huge polar energy component (ΔGGB = 240.3 kcal/mol) in the binding process of the multi-epitope vaccine polypeptide. Therefore, the nonpolar energy was known as the main driving force in the vaccine binding to TLR-3, and this hydrophobic contribution leads to a thermodynamically favourable interaction (∆Gbinding = -138.11 kcal/mol). To further clarify the binding mode, the binding free energy of the protein complex was broken down into residue-residue pairs through the binding free energy decomposition analysis.

According to the data, there are several residues of vaccine polypeptide with less than −1.5 kcal/mol free energy of contribution in the binding mechanism. The binding pose of vaccine with the key residues are illustrated in Fig. 5.

Table 2. Binding free energy calculation for the multi-epitope vaccine candidate-TLR-3 complex

	Energy values (kcal/mol)
∆Eele^a	-241.5 +/- 14.81
∆EvdW^b	-118.81 +/- 9.0
∆GGB^c	240.3 +/- 16.71
∆GSA^d	-18.11 +/- 1.1
∆Epolar^e	-1.2 +/- 0.4
∆Enon-polar^f	-136.92 +/- 4.07
∆Gbinding	-138.11 +/- 9.46

a Electrostatic contribution, ^b van der Waals contribution, ^c Polar contribution of the solvation effect, ^d Non- polar contribution ofsolvation effect, ^e ∆Epolar = ∆Eele + ∆GGB, ^f ∆Enon-polar = ∆EvdW + ∆GSA.

Since the beginning of SARS and MERS epidemics, respectively in 2002 and 2012, until very recently, several research reports and patents have concerned the design of appropriate vaccines16-21. Nonetheless, no specific vaccine is presently available in the markets for prevention against these respiratory infectious diseases17,22. This implies an urgent demand for changing the paradigms in the case of COVID-19. Research on COVID-19 prevention was begun immediately after release of relevant basic data such as its full genomic sequence. Specific epitope regions in SARS-CoV-2 with high homology to SARS-CoV, the best-characterized coronavirus in terms of epitope responses, were identified23-25. Antigenic properties of spike glycoprotein were more focused by theory and experimental researchers26-28. A multi-epitope design based on S-protein was proposed29. Despite the current highly active research community as well as the efforts in the industry section in the road to find an optimal immunogenic formulation, the extreme diversity in available design choices with no a priori image of their effectiveness is the fundamental obstacle in this route. To overcome this issue, mass design of various vaccine species and development of appropriate screening methods can be proposed as a solution. To date, limited number of designs have been reported. This research offered a first step in designing an alternative candidate for developing a COVID-19 vaccine, and aims to instigate further exploration of design and screening strategies.

Deployment of classic live or attenuated vaccines is associated with biosafety issues such as autoimmune or strong allergic responses, plus difficulties with their synthesis and manufacture.

These drawbacks might be addressed by developing fully synthetic peptide-based vaccines30. Integrating multiple immunodominant sites of viral pathogens in the vaccine structure helps augment the antigenic effect. In this study, the amino acid sequence of the whole set of structural proteins of SARS-CoV-2 was included for epitope identification. There is much evidence on immunity-related advantages of each of the S, E, M and N proteins of SARS-COV or SARS- CoV-223,31-34. This choice, besides, aimed to cope with potential vaccine side effects. Antibody- dependent enhancement of infectivity (ADEI) is one great challenge of subunit vaccines, which refers to the reduced specificity in response elicitation because of the numerous conformations adopted by peptide vaccines10. Strategies to mitigate this concern include: 1- immunofocusing by considering several most antigenic epitopes35, 2- inclusion of structural proteins: structurally flexible coronaviral epitopes may be of limited value for in vivo immunotargeting and require to be replaced by conserved epitopes with low structural plasticity6. Structural proteins are characterized by highly conserved sequences, thus the linear epitopes remain highly stable in these proteins and the conformational epitopes preserve their structural patterns during various steps of the virus cycle. Our choice to include the full set of SARS-CoV-2 structural proteins has thus combined the advantage of augmented immunogenicity with both strategies proposed for reducing ADEI.

Reports revealed that multiple antigenic peptides induce stronger B and T cells immune responses than un-conjugated peptide epitopes36-38. Thus, consecutive sequences of HTL and CTL epitopic peptides were fused to each other using accepted linkers. The T-cell and B-cell binding, and IFN-γ inducing potential of epitopes were then identified.

Epitope-based peptide vaccines induce relatively weak immune response, when used alone19. The immunoreactivity could be improved by adding proper adjuvants to this type of vaccines.

The vaccine construct in this study was prepared by fusing the epitopic peptides to an immunogenic adjuvant. Use of appropriate adjuvants has also been shown to help induce durable IFN-γ responses; furthermore, lung eosinophilic immunopathology, though less indicated for subunit vaccines, can be avoided by administration of TLR agonist adjuvants6. In this study, the binding of the built vaccine model with its specific immune receptor TLR-3 was validated by performing molecular docking, MD simulation and free energy calculations. While we note that other Toll-like immune receptors such as TLR-2, -4 and -5 may be activated by COVID-19 virus, they were not considered here as their exact roles are unclear with some functioning even to the advantage of the coronavirus39.

In the ongoing urgent situation brought about by SARS-CoV-2, it is hard to fast counteract the circulating disease through preventative or therapeutic measures. The multi-epitope-based subunit vaccine design presented in this study can be promising, as it incorporates a priori bioinformatics predictions and up-to-date immunological knowledge. By proposing this vaccine polypeptide, this study also hopes to encourage a rapid design and screening strategy to find one formulation with the highest immunogenicity and biosafety.

SARS-CoV-2 structural protein sequences. The amino acid sequences of SARS-CoV-2 structural proteins, including the spike glycoprotein (S), envelope protein (E), membrane protein (M), and nucleocapsid phosphoprotein (N), were retrieved using the NCBI reference genome of the virus (accession number NC_045512.2).

Identifying cytotoxic T lymphocyte (CTL) epitopes. Predicting peptides that are capable of inducing CTL responses is a crucial step in the design of epitope-based vaccine. The MHC-І binding tool of Immune Epitope Database and Analysis Resource (IEDB; http://tools.iedb.org/mhci) was used to predict the CD8+ T-cell epitopes borne in S, E, M and N proteins12,40,41. In this step, the ANN 4.0 method was set as the prediction method. The human was selected as the source species. The maximum IC50 value was set to 50 nM, and percentile rank < 1 was considered since lower score indicates high affinity42,43.

Identifying helper T lymphocyte (HTL) epitopes. IEDB (http://www.iedb.org) was used to predict MHC-II binding of 15-mer epitopes for viral structural proteins against human HLAs such as HLA-DRB1*15:01, HLA-DRB4*01:01, HLA-DRB3*01:01, HLA-DRB5*01:01, HLA-DRB1*03:01, HLA-DRB3*02:02 and HLA-DRB1*07:01 (Supplementary Table S8 online), using NN-align 2.3 method^13,14,44,45. Epitopes with the IC50 values < 50 nM have high affinity. The maximum value of 500 nM and 5000 nM indicate intermediate and low affinity of epitopes, respectively⁴⁶. The 15-mer epitopes with IC50 values < 50 nM were considered for next analysis46-48.

B-cell epitopes prediction. The ABCpreds server was used to predict 16-mer linear B- lymphocyte (LBL) epitopes with a threshold of 0.5149. In addition, the ElliPro tool of IEBD was utilized to predict conformational and linear B-cell epitopes of the vaccine polypeptide.

Assessment of identified epitopes for antigenicity, allergenicity, and toxicity. The antigenic potential of each of the T and B cells epitopes was predicted by VaxiJen v2.0 with a threshold of 0.450. The predicted T and B cells epitopes were then further evaluated to check their toxicity and allergenicity, with ToxinPred and AllergenFP v1.0 server, respectively51,52. Then, the ability of each of the HTL and B cell epitopes (CD4+) to induce the secretion of cytokines, such as interferon-gamma (IFN-γ), interleukin-4 (IL-4) and interleukin-10 (IL-10), to overcome the inflammatory response and prevent tissue damage was predicted by IFNepitope, IL4pred and IL10pred server tools, respectively53-55. The IL4pred and IL10pred operations were carried out based on SVM method and hybrid method respectively, with other parameters kept as default12,13.

Designing the multi-epitope vaccine polypeptide construct. To develop a multi- epitope vaccine, we selected those predicted epitopes with high antigenic potential, which were not identified as allergic or toxic, and had good solubility when highly expressed. To construct the vaccine polypeptide, the selected CTL, HTL and LBL epitopes were fused together using appropriate linkers56,57. An adjuvant was also considered to increase the immunogenic capacity of the multi-epitope vaccine12,46. Accordingly, a 45-amino acid sequence prepared from β-defensin-2 protein was added to the N terminus of the vaccine sequence using the EAAAK linker57.

Immunogenic, allergenic and physiochemical evaluation of vaccine construct. The antigenicity of the multi-epitope vaccine polypeptide was predicted utilizing the VaxiJen v2.0 tool, with the threshold value 0.450. The allergenicity of the vaccine was analysed using AllerTOP v.2.0 and AllergenFP v.1.0 servers51,58. The ProtParam server was employed to evaluate the physical chemistry properties of the vaccine construct, such as amino acid composition, molecular weight, theoretical isoelectric point (pI), grand average of hydropathicity (GRAVY), aliphatic and instability index, and in vitro and in vivo half-life59.

Vaccine polypeptide structure modelling, refinement and validation. The SOPMA server was applied for analysing the secondary structural properties of the multi-epitope vaccine polypeptide60. The GalaxyWEB server was employed for modelling and refinement of the 3D structure model61. The server relaxes the model structure using repacking and molecular dynamics (MD) simulation. Next, RAMPAGE server and ProSA-web were used to validate the refined 3D model62,63. All of these tools give us the overall quality of the 3D structure of the peptide vaccine.

Molecular docking of the vaccine polypeptide to TLR-3. The binding of antigenic agents with the target immune cell protein is crucial for the creation of a suitable immune system response. For analysing the binding pattern of the multi-epitope vaccine polypeptide with TLR-3 (PDB ID: 2A0Z)64, molecular docking analysis was performed by Hdock, Zdock, Cluspro and Hawkdock65-68. Among the molecular species docked by each of these applications, the best outputs were extracted, followed by the four docked molecules uploaded into the Hawkdock program, and the free energy of binding of each complex calculated. Based on this screening, it was determined that the model selected from the Cluspro output had the best binding free energy. Therefore, this molecular species was chosen as the primary structure for initiating the molecular dynamics (MD) simulation.

MD simulation of the vaccine-receptor complex. To determine the structural stability and to study the molecular details of the interactions between TLR-3 and the multi-epitope vaccine polypeptide in the docked conformation, MD simulation was performed. Briefly, the system including vaccine polypeptide-TLR-3 was simulated by the Gromacs-2020 package applying OPLS-AA force field69. The complex system was solvated using TIP3P water model. Then, the genion module was utilized to neutralize the whole system. Next, the conjugate gradient algorithm was applied to minimize the energy of the system. In the NVT ensemble, the temperature of the system gradually increased from 0 to 310 K during 400 ps. Subsequently, in the NPT ensemble, 500 ps simulation was carried out at a pressure of 1 atm and a temperature of 310 K. Production simulation for 24000 ps was then implemented. The particle-mesh Ewald (PME) and the LINCS algorithms were applied to assess all electrostatic connections and to restrain all bond lengths in the protein, respectively. Moreover, periodic boundary condition was utilized during the simulation. The final coordinates obtained for the complex system were analysed with classic MD analyses, plus the MMPBSA method for calculating the free energy of intermolecular interactions70. The results were visualized by Pymol (Schrodinger L.L.C.).

Author Contributions

EB (First author) performed data curation, analysis, validation and visualization; and wrote the original draft. BS contributed in resource and funding acquisition, and writing the original draft. AN contributed in project administration, resources and funding acquisition, and validation. EB (Last author) conceptualized the work, defined methodology, and contributed in project administration, supervision, visualization, completing the draft. All authors reviewed the final manuscript.

Additional Information

Supplementary information accompanies this paper including Supplementary Tables S1-S8 and Figures S1-S3.

Competing Interests Statement: The authors declare no competing interests.

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Immunoinformatic design of a COVID-19 subunit vaccine using entire structural immunogenic epitopes of SARS-CoV-2

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