Immunoinformatic approach to design a multiepitope vaccine targeting non-mutational hotspot regions of structural and non-structural proteins of the SARS CoV2

Introduction

Immuno-pathogenesis of the infectious epidemic COVID-19 emerged as serious intimidation worldwide. In the Coronaviridae family, among four different coronavirus classes (alpha, beta, delta and gamma), alpha and beta positive-sense RNA virus strains have been confirmed for broadly epidemic infection (De Wilde et al., 2018; Tahir ul Qamar et al., 2020). Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV2) mediated outbreak of the disease was firstly reported in Wuhan city, China, in December 2019 (Gorbalenya et al., 2020; Huang et al., 2020; Perlman, 2020; Tahir ul Qamar et al., 2019; World Health Organization, 2020; Wu, Leung & Leung, 2020; Zhu et al., 2020). The outbreak has so far infected more than 10,00,000 patients worldwide on dated 10 May 2020. SARS CoV2 genome sequence comparison showed almost 96%, 79.5% and 40% similarities with bat coronavirus, SARS CoV and MERS CoV strain, respectively (Alamri, Tahir ul Qamar & Alqahtani, 2020; Benvenuto et al., 2020; Zhou et al., 2020). The clinical symptoms of SARS CoV2 exhibit up to 14 days in infected people with fever (≥38 °C), diarrhea, dry cough, low peripheral WBC count, respiratory disorder and low lymphocyte count (Huang et al., 2020).

Among SARS CoV2 genomic RNA, 66% codes for the non-structural or essential proteins, and 33% codes for the structural or accessory proteins. Non-structural proteins have a significant role in virus replication and transcription in the host cell, while structural proteins cover the outer surface and participate in the host evasion system to cause the infection. SARS CoV2 mutation prone (Jiayuan et al., 2020) genomic organization is a difficult task to develop a vaccine that is composed of 5′-leader-UTR-replicase-ORFab-S(Spike)-E(Envelope)-M(Membrane)-ORF6-ORF7a-ORF8-N(Nucleocapsid)-3′UTR-polyA tail. Accessory proteins like ORF3a, ORF7a, and ORF8 are essential for viral pathogenesis (Seema, 2020; Zhu et al., 2020).

To overcome the issues such as cost and time associated with traditional method (monoclonal oligonucleotides, small drug molecules), of disease prevention (Li & De Clercq, 2020), the virus was analyzed by computational methods (Chen, Liu & Guo, 2020). Recently published immuno-informatics based vaccine design against MERS virus, Ebola virus chikungunya, and Zika showed the promising potential to fight against disease (Ahmad et al., 2019; Shahid et al., 2020; Tahir ul Qamar et al., 2018, 2019). Reverse vaccinology includes different software algorithms to evaluate the immunological data that analyze the epitopes binding efficiency with human leukocyte antigen (HLA) alleles, antigenicity, allergenicity and toxicity to design the potential multiepitope subunit vaccine (De Gregorio & Rappuoli, 2012; Khan et al., 2018; Mirza et al., 2016; Patronov & Doytchinova, 2013). The final multiepitope vaccine is a group of different epitopes joined with the help of linkers that may enhance specific adaptive-immune responses in the host cell (Brennick et al., 2017; Chauhan et al., 2019; Jensen & Andreatta, 2018; Lu & Meng, 2017; Nain et al., 2020; Purcell, McCluskey & Rossjohn, 2007; Saadi, Karkhah & Nouri, 2017). To activate the host immune system, interaction of toll-like receptors (TLRs) with the designed vaccine could be a potential approach to combat viral infection. Intracellular TLRs that present on cell endosomes interact with ssRNA (TLR-7 and TLR-8), dsRNA (TLR-3) and CpG DNA (TLR-9) to activate the NFkβ mediated immune/cell response (Carty & Bowie, 2010). With protein-based epitope vaccine designing, TLR-2, TLR-4, TLR2/6 and TLR1/2 heterodimer mediated immune cell activation (IRF3/7 and NFKβ) is an essential factor to activate the innate immunity. Basal TLR-2 and TLR4 activation needs for host protective immunity, whereas its overexpression leads to subvert effect by inducing the viral cell replication in the host cell (Olejnik, Hume & Muhlberger, 2018); hence, it is better to target TLR1/2 heterodimer mediated activation signaling with mild TLR-4 interaction/activation (Olejnik, Hume & Muhlberger, 2018).

In the present study, non-mutational sequences of SARS-CoV2 were targeted to design a multiepitope vaccine construct. SARS-CoV2 proteome was investigated to identify antigenic proteins and different T-cell and B-cell epitopes were identified in selected proteins, and their antigenicity, allergenicity and physiochemical properties have been evaluated. The molecular docking of final epitope constructs was performed with different TLRs and HLA alleles to confirm the stable binding interaction of the multi-epitope vaccine-receptor complex. Vaccine constructs were further assessed for its cross-reactivity and immune response in humans, as well as their similarity with the human microbiome.

Materials and Methods

Result

Analysis of protein antigenicity and trans-membrane helicity

The workflow has been discussed in Fig. 1, which explains the reverse vaccinology mechanism used in the present study. Amino acid sequences of target proteins were collected in FASTA format to analyse antigenicity and trans-membrane helicity. All the proteins with their antigenicity score such as nucleoprotein phosphoprotein (0.50), ORF10 protein (0.71), ORF8 protein (0.65), ORF7a protein (0.64), ORF6 protein (0.61), membrane glycoprotein (0.51), an envelope protein (0.60), ORF3a protein (0.49), surface glycoprotein (0.46) and ORF1ab protein (0.46) showed their potential to exceed the immune response in the host cell. To elicit the host cell’s immune response, the transmembrane helicity of viral proteins has been evaluated. With the help of TMHMM and Protter servers, we have identified the extracellular, transmembrane and cytosolic peptides. The protein protter results showed that nucleoprotein phosphoprotein, ORF6 protein, and ORF10 protein are completely cytosolic. Hence, for further study, these three proteins were eliminated, and the rest of the seven proteins were considered for further study in designing the chimeric vaccine against SARS CoV2. The extracellular peptides of these proteins would help to interact with host PAMPs and maximize the solubility of designed vaccines.

Analysis of physicochemical properties of selected vaccine construct VTC3

Designed vaccine constructs VTC1, VTC2 and VTC3 (Table 7), showed antigenicity with no allergenicity and no toxicity. The physiochemical analysis of highly antigenic vaccine construct VTC3 showed that it has 227 amino acids, a molecular weight of 24 kDa, and an instability index of 23.36 that represent the protein’s stable nature. VTC3 construct contains the aliphatic and GRAVY index value of 68.72 and −0.455, respectively. PESIPRED secondary structure analysis of vaccine construct VTC3 showed the 62.56% alpha helicity, 7.05% extended strands, 5.73% beta-turn with 24.67% random coils. VTC3 tertiary structure has been modeled, refine, and validated by Ramachandran plot that showed 97% residue in the favored region (Fig. 2). The model has a Z-score of −3.35 and QMEAN of −10.75 (Fig. S1). To validate Phyre 2 model, the ab initio model was generated using RaptorX. Both models are similar, and the Ramachandran plot of the RaptoX model showed 99% residue in the favored region. This confirms that phyre2 model is valid hence used for further study.

Table 7:

Sequence, Antigenicity, and Allergenicity of designed multi-epitope Vaccine constructs.

S. no	Name	Vaccine construct	Antigenicity	Allergenicity
1	VTC1	EAAAKCTQHQPYVVDDPCPIHEYGAEALERAGYSRVKNLNSSRVPGGGSMRIFTIGTVHEYGAEALERAGKTQSLLIVNNATNVVGGGSLKQGEIKDATPSDFVRHEYGAEALERAGWTAGAAAYYGGGSLSFELLHAPATVCGPHEYGAEALERAGVSGTNGTKRFGGGSLSDDAVVCFHEYGAEALERAGKSAFYILPSIISNEKGGGSVVKIYCPACHNSEVGPEAAAK	0.51	Non-allergenic
2	VTC2	EAAAKCTQHQPYVVDDPCPIHEYGAEALERAGYSRVKNLNSSRVPEAAAKLKQGEIKDATPSDFVRHEYGAEALERAGMRIFTIGTVEAAAKKTQSLLIVNNATNVVHEYGAEALERAGDPFLGVYYHKNNKSWMESEFRVYSSAEAAAKWTAGAAAYYHEYGAEALERAGPDPSKPSKREAAAKNYIFWRNTNPIQLSSHEYGAEALERAG SSEIIGYKAIDGGVTREAAAK LQAENVTGL	0.47	Non-allergenic
3	VTC3	EAAAKGCSCDQLREPMLQSADAQSHEYGAEALERAGFELEDFIPMEAAAKQMNLKYAISAKNRARHEYGAEALERAGEVRQIAPGQTGKIADYEAAAKLSFELLHAPATVCGPHEYGAEALERAGWTAGAAAYYEAAAKLKQGEIKDATPSDFVRHEYGAEALERAGMRIFTIGTVEAAAKYSRVKNLNSSRVPHEYGAEALERAG PYVVDDPCPIHFYSKWEAAAK	0.61	Non-allergenic

DOI: 10.7717/peerj.11126/table-7

Molecular docking analysis confirms the interaction of VTC3 with TLR and HLA

To be effective against SARS-CoV2, a vaccine should have the capacity to activate the immune response of the human host. The virus can subvert the host protein function, which plays a role in host cell invasion or virus persistence. In a previous study, it has been shown that TLRs have an extensive role in pathogen persistence and clearance. Hence, docking analysis was performed, which showed that our construct VTC3 interacts with TLR1/2 complex, TLR1, TLR3, TLR4, TLR6 but has the highest binding affinity with TLR1/2 complex, followed by TLR4 and TLR6 (Table 8). The docked poses are shown in Fig. 3. As mentioned in the introduction, the interaction of VTC3 with TLR1/2 is important for the immune response for viral infection; hence docking results further confirm it. In addition to that, molecular docking of VTC3 with different HLAs (MHCs) has been performed, which was important to induce MHC-I and MHC-II to activate the immune response in the host cell. Best docked TLRs and HLA alleles molecules were further confirmed by the HDOCK server (Fig. S2). The binding affinity and dissociation constant of VTC3 were favorable with HLA, TLR1/2 and TLR4 (Table S1).

Table 8:

Molecular docking of vaccine construct with different TLRs and HLA alleles.

S. no.	Receptor	Receptor PDB	Global Energy
1	TLR4	2Z62	−1.64
		3UL8	2.16
		2Z63	−28.76
		3UL9	−1.43
		2Z65	−18.55
		2Z66	−23.62
		3FXI	−6.93
		3ULA	−1.75
		4G8A	−2.65
		5NAM	−6.18
		2Z64	1.23
2	TLR1-2 hetero dimer	2Z81	−0.88
		2Z80	−31.20
		2Z82	0.22
		2Z7X	3.75
3	TLR1	6NIH	−1.95
		1FYV	−6.49
4	TLR2	6NIG	10.81
5	TLR6	3A79	−13.05
6	TLR3	2A0Z	−8.85
		1ZIW	6.11
7	HLA Alleles	5IM7	−24.66
		1XR8	−1.95
		1SYS	−17
		1A6A	−5.59
		1A1M	−3.24
		1BX2	6.09
		1H15	−9.32
		1ZSD	5.51
		3C5J	5.12
		4O2E	9.68

DOI: 10.7717/peerj.11126/table-8

MDS analysis confirms the strong interaction of VTC3 with TLR1/2 heterodimer and HLA

Binding affinity from the PatchDock result showed that VTC3 has better interaction with the TLR1/2 complex than TLR4, while Binding affinity from HDOCK result showed TLR4 have better interaction than TLR1/2; hence both complexes were analyzed by MDS analysis. MDS was performed till 10 ns for both the complex, and results were analyzed for RMSD, RMSF, etc. RMSD calculation showed that the VTC3-TLR1/2 complex is stable throughout simulation with RMSD of around 3 Å (Fig. 4A), and the VTC3-TLR4 was found to be relativity less stable with RMSD of 7 Å (Fig. 4C). Similarly, the RMSF analysis showed that the VTC3-TLR1/2 complex has less fluctuation with RMSF of around 3 Å (Fig. 4B) while the VTC3-TLR4 complex showed that the vaccine has an RMSF of 7 Å (Fig. 4D). MDS result suggests that multiepitope vaccine construct VTC3 showed a stronger and stable interaction with TLR1/2 complex compared to TLR4 (Fig. 4); this indicated that this vaccine construct induces an immune response suitable for clearance of SARS CoV2. For a vaccine construct, it is also important to interact with HLA. MDS analysis of the VTC3-HLA complex was investigated till 50 ns, and the result was analyzed for RMSD and RMSF. RMSD calculation showed that the VTC3-HLA complex is stable throughout simulation with RMSD of around 5 Å at 50 ns (Fig. 5), and RMSF analysis showed that the VTC3-HLA complex has some fluctuation at the terminal. Both the data suggest the interaction between VTC3 and HLA.

Mutational profiling of multiepitope vaccine VTC3 showed less mutation sensitivity

Mutational sensitivity profiling of the VTC3 vaccine construct (Fig. 6) showed that only a few epitope residues of VTC3 have ΔΔG_pred > 0, suggesting that vaccine construct VTC3 has less mutation sensitivity, which enhances the possibility of vaccine constructs to be effective. Similarly, analysis of vaccine construct VTC3 in the CoV-GLUE database showed less mutation hot spots in our vaccine construct (Table S5). This database contains all replacements, insertions and deletions, which have been observed in the GISAID hCoV-19 sequence sampled from the pandemic. Interacting residues of vaccine construct VTC3 with the HLA, TLR1/2 complex, TLR4 has been identified. The result showed that most of the residues of VTC3 with the mutation do not involve the physical interaction with these proteins. These results further support the efficacy of the vaccine construct.

Immune simulation confirms the strong immune response against the VTC3, as well as virus protection

In the presence of antigen, the C-ImmSim model illustrates the host immune responses at both the humoral and the cellular level. The immune response against the VTC3 vaccine showed an appropriate immune response and virus antigen elimination (Fig. 7). Injection of the vaccine VTC3 without LPS shows elevated IgM level after the first injection. Its concentration further increases along with IgG1 after subsequent injection of VTC3, showing secondary and tertiary immune response. The secondary and tertiary response eliminates the antigen on a shorter timescale. It is also observed that when the virus was injected at 24 weeks (at 168 days), it is eliminated at the shortest timescale that showed the presence of memory cells to react with virus antigen. It was also observed that after virus injection, IgG1 titer is more than IgM titer (Fig. 7A). Similarly, B-cell population increases with subsequent injections of VTC3 that show a typical immune response. The memory B cell also increases after second injection and remains almost similar (Fig. 7B). The population of Th cells increases after injection of VTC3 and Th memory increase after second injection and remains almost same afterward (Fig. 7C). Among the Th cells, the population of Th1 is more dominant. After injection of VTC3, the cytotoxic TC population increases and remain constants after first injections with a TC memory (a.u. 900). It was observed that TC memory was further enhanced after injection of the virus at 168 days. It was also observed that VTC3 injection increases the IL-12 level and IFN-gamma showed a potent innate and adaptive immune response. The population of macrophages also increases after the injection of VTC3. Other cells like the dendritic cell, natural killer cells and epithelial cell population, plasma B lymphocyte population also support immune reaction. In brief, the result explains the development of strong immune memory with subsequent virus eliminating potential.

Codon adaptation and in silico cloning

The Java Codon Adaptation tool converted the protein sequence of VTC3 into cDNA sequence. This cDNA sequence contains 52% GC content and CAI value of 1.0. This high CAI value shows a high expression of the vaccine gene in the host cell. The final vaccine construct (VTC3) was cloned into a pET28a vector between the HindIII and BamHI (Fig. 8).

Discussion

In the present study, a multiepitope vaccine constructs VTC3 for SARS CoV2 have been proposed with the help of reverse vaccinology that compiles the outer surface-exposed epitopes of the structural and non-structural proteins of this virus. There are different therapeutic strategies, and ongoing research has been tried to control SARS CoV2 (Felsenstein et al., 2020; Tiwari, 2020b, 2020a). Although different groups have tried to design a vaccine against SARS-CoV2, the present study reports a comprehensive approach to design a vaccine construct that taking care of all possible design shortcomings such as absence of surface epitopes, inappropriate TLRs interactions, less population coverage, interaction with the human microbiome, presence of cross-reactivity with human proteins, epitope form mutation hot spots and absence of immune response, etc. SARS CoV2 structural proteins like surface glycoprotein, membrane and envelope protein have a significant contribution in surface adherence and internalization, while non-structural protein is virulence-associated factors that cause immune-pathogenesis. SARS CoV2 host cell internalization is facilitated by surface glycoprotein (spike protein) interaction with the ACE2 receptor. Targeting the outer exposed peptides of both structural and non-structural protein as a vaccine target would be a promising approach to induce both humoral and cellular immune response that recognizes the virus and killed it. These proteins were shortlisted by antigenicity score and trans-membrane helicity. The shortlisted proteins were used to identify surface-exposed peptides. Surface exposed peptides of these proteins were further analyzed to determine MHC I, II and B cell-mediated epitopes. The selected epitopes were shortlisted by their antigenicity, allergenicity, toxicity, cross-reactivity, immunogenicity and IFN gamma secretion scores. Cross-reactivity of selected epitopes of the virus with the human’s protein has also predicted that eliminates autoimmune reactivity for the vaccine construct. These epitopes were used to design different vaccine constructs (VTC1, VTC2 and VTC3) with the help of linkers. Antigenicity, allergenicity and physiochemical analysis of vaccine constructs were further determined to enhance the peptide potency. For designing the reliable vaccine, a mutational hot spot site has been analyzed, and found that epitopes belong to the non-mutation site of the protein. To minimize similarity with human proteome and gut microbiome, BLASTP analysis has been done that showed no similarity of VTC3 construct with host cell proteins.

The finalized construct VTC3 should have the potential to interact with pathogen-associated molecular patterns, activates the host’s innate immune system, and consonance of the adaptive immunity via TLRs (TLR1, 2, 3, 4, 6, 7, 8 and 9). It has been observed that the TLRs mediated virus interaction not-only combat virus virulence and infection but also sometimes initiate the host system to overturn downstream signals for the benefit (replication and survival) of the virus-cell. During the viral infection, membrane-associated proteins interaction with TLR2 and TLR4 plays a nuanced role in exceeding inflammatory responses via adhesion and invasion that vitiate the host cell (Olejnik, Hume & Muhlberger, 2018). Simultaneously with TLRs, HLA alleles have a significant contribution to the activation of the robust immune response. The strong HLA-epitope interaction would maintain the signaling cascades to activate the immune response for the viral infection clearance. To prove the modeled VTC3 construct, we have docked it with different HLAs and TLRs. Presently, the focus lies on the development of vaccines against viruses that can activate the innate immune system via TLR1/2 (Carty & Bowie, 2010; Dowling & Mansell, 2016; Jensen et al., 2018) to overcome the subvert effect of the virus on TLR mediated signaling. It is reported that the CD8⁺ T cell-mediated HLA allele (HLA-B*5801, PDB ID 5IM7) unique interaction with immune dominant peptide contributes as a potential to control and prevent viral infection (Li et al., 2016). The interaction of VTC3 was investigated with molecular docking and MDS, and the result is in agreement that the VTC3 construct has the highest affinity with allele HLA-B*5801. It might also create a ray of hope for the potential creation of vaccines or convalescent serum antibodies against SARS CoV2. Vaccine behaviors in human is a significant concern hence immune simulation was performed that shows strong immune response after injection of VTC3, and virus elimination. The designed multiepitope vaccine construct VTC3 should be tested experimentally for therapeutic potency in future studies. Experimental validation is necessary to demonstrate the potency of the designed vaccine in future studies.

Conclusion

The high mortality and morbidity rate of COVID 19 is unprecedented due to the unavailability of vaccination; hence effective treatment strategies (inhibitor, drugs, or vaccine), rapid development, trials and production are needed immediately for this global pandemic disease. To reach out the success, it is necessary to evaluate all possible vaccine candidates to find out the one viable outcome. To increase the chance of success, WHO initiated vaccine solidarity trials to test all vaccine candidates until they fail. In the present study, an attempt was made to design a multi-epitope vaccine against SARS CoV2 that targets its exposed peptides of structural and non-structural proteins. Immunogenicity, allergenicity, toxicity and potent IFN-gamma inducer scores have also been analyzed to narrow down efficient epitopes further. Peptide matching with the human proteome showed no indication of possible cross-reactivity. However, current reverse immuno-informatics approaches were executed to target surface-exposed proteins for enhancing effective host innate with humoral and cellular immune responses. The present study concludes the design of VTC3 as a multiepitope vaccine against SARS CoV2.

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