key: cord-0992091-7t45osdc
authors: Naveed, Muhammad; Tehreem, Sana; Arshad, Sundas; Bukhari, Syeda Aniqa; Shabbir, Muhammad Aqib; Essa, Ramsha; Ali, Nouman; Zaib, Sumera; Khan, Ajmal; Al-Harrasi, Ahmed; Khan, Imtiaz
title: Design of a novel multiple epitope-based vaccine: An immunoinformatics approach to combat SARS-CoV-2 strains
date: 2021-05-04
journal: J Infect Public Health
DOI: 10.1016/j.jiph.2021.04.010
sha: 0a1c98e1591ba5e41af93149e15e727b7d917da4
doc_id: 992091
cord_uid: 7t45osdc

Background Since the SARS-CoV-2 outbreak in December 2019 in Wuhan, China, the virus has infected more than 135 million individuals across the world due to its human-to-human transmission. The USA is the most affected country having more than 58 million cases till date. Sudden high fever, pneumonia and organ failure have been observed in infected individuals. Objectives In the current situation of emerging viral disease, there is no specific vaccine, or any therapeutics available for SARS-CoV-2, there is a dire need to design a potential vaccine to combat the virus by developing immunity in the population. The purpose of present study was to develop a potential vaccine by targeting B and T-cell epitopes using bioinformatics approaches. Methods B and T-cell epitopes prediction from novel M protein-SARS-CoV-2 for the development of a unique multiple epitope vaccine applying bioinformatics approaches. These epitopes were analyzed and selected for their immunogenicity, antigenicity scores, and toxicity in correspondence to their ability to trigger immune response. In combination to epitopes, best multi-epitope of potential immunogenic property was constructed. The epitopes were joined using EAAAK, AAY and GPGPG linkers. Results The constructed vaccine showed good results of worldwide population coverage and promising immune response. This constructed vaccine was subjected to in-silico immune simulations by C-ImmSim. Chimeric protein construct was cloned into PET28a (+) vector for expression study in Escherichia coli using snapgene. Conclusion This vaccine design proved effective in various computer-based immune response analysis as well as showed good population coverage. This study is solely dependent on developing M protein-based vaccine, and these in silico findings would be a breakthrough in the development of an effective vaccine to eradicate SARS-CoV-2 globally.

Coronaviruses are enveloped RNA viruses that are known to cause various diseases including respiratory, enteric, hepatic and neurological in humans, birds and other mammals [1] . There are six species of coronavirus having capability to cause disease in humans and four viruses HKU1 OC43, NL63, and 229E are known to cause cold like symptoms in humans [2] . In past, though an unknown coronavirus has already caused an outbreak of SARS which is also known as Severe Acute Respiratory Syndrome in humans in 2003 [3, 4] . In December 2019, an enormous number of pneumonia cases were observed in Wuhan, China. On 2 Jan, 2020, 41 patients were diagnosed with infection of a novel coronavirus (SARS-CoV-2) in China [5] . Later, on March 11, 2020, WHO announced the COVID-19 outbreak as pandemic. Most of the infected people were exposed to the wet animal market of Wuhan, China where the animal meat was sold routinely.

So, it was suggested that the virus may belong to zoonotic origin [6] . However, cases were also reported in the people who were not exposed to wet animal market. On the basis of this, new reports suggested that the actual route of virus transmission is from person to person [7] . The detail analysis showed that the virus may spread from person to person through contact and the droplet transmission i,e., from the infected individuals by coughing and sneezing [8] .

The WHO stated to get extensive measures in order to control the spread. For this purpose, isolation of infected individuals is recommended, and traveler screening is getting mandatory to avoid further spread of the infection. Although, early death cases were observed in elder people because of their weaker immunity allows the viral replication hence the progression of viral infection occurs [9] . The latency periods of virus is almost 3 to 7 days according to epidemiological studies with a maximum of 14 days [10] . The novel coronavirus (SARS-CoV-2) is contagious during the latency period and promised to be emerging continuously at rapid rate.

This property of the novel virus makes it more lethal and difficult to combat. The COVID-19 infection shows common symptoms like flu including fatigue, fever, and dry cough. In some cases, lymphopenia as well as acute respiratory symptom are observed. However, about 30% severe cases of the disease are held in intensive care, with median mortality rate of 4.3% [11] .

The positive sensed, non segmented single stranded RNA genome of SARS-CoV-2 is responsible for making many proteins for its survival and attack. It has four main structural J o u r n a l P r e -p r o o f proteins such as Envelope glycoprotein (E), Spikes glycoprotein (S), Matrix/membrane glycoprotein (M) and nucleocapsid (N). For the selection and entry into the target cell, the novel coronavirus SARS-CoV-2 uses its spike proteins. Crown-like shape of the spikes protein forms homotrimer onto the viral surface and helps in binding of viral body onto the ACE2 receptor of the host cell. When coexpressed, the instinctive envelope trimeric S glycoprotein is incorporated onto virus like particle in order to cause infection [12] . According to recent reports, a protein on host surface, cellular protease TMPRSS2 is used for SARS-CoV-2 priming [13] . Therefore, the diagnosis of lethal pathogen SARS-CoV-2 can be done by various methods including RT-PCR and CT-scans [14] . CT scan findings are considered better and authentic than RT-PCR diagnostics due to its good sensitivity power. The sensitivity of CT-scan was observed as 97.2% as compared to RT-PCR (83.3%). So patients with promising CT-scan findings and negatively reported on PCR should be isolated [15] .

Although at present, there is no authentic treatment method available for the novel coronavirus disease . But some reported drugs are having a noticeable effect over the patients [16] . Remdesivir is observed to be an effective on coronavirus patients due to promising effects.

Remdesivir is an adenosine correspondent which causes pre-mature termination by integrating into nascent viral RNA chains. Hydroxychloroquine, another drug, is recommended to be used in the updated version of guidelines due to its acceptable safety and efficacy [16] . However, a parasitic drug ivermectin, is now declared FDA-approved drug to be an inhibitor of COVID-19 by showing almost 5000 folds reduction in viral replication in many in-vitro studies [17] . Since there is a large work in the pharmaceutical field, still not a single drug cures this disease completely. This arises the need of vaccine development in order to target virus at its molecular level. However, some advancement in the field of vaccine development has been made. Nano based vaccination and homology based vaccination against coronavirus are new talks of the era.

On the other hand, recent advancement at vaccine strategies like live-attenuated vaccine, protein subunits vaccine, inactivated vaccine, viral vector vaccine also facilitates in encouraging advancements [18] . The role of promising vaccine will be in activation of initiate herd immunity.

Herein, we chose M protein or vaccine design, due to its high antigenic and conserved property, for the purpose of multi-epitope vaccine development of the novel coronavirus (SARS-CoV-2).

As M protein is a main component of viral envelope, it cotributes in viral morphogenesis and interaction during assembly with other cell bodies. Hence, it plays a central role in viral J o u r n a l P r e -p r o o f

immunoenvasion. This novel vaccine shows potential results by accomplishing all-natural criteria in the artificial immune environment. The vaccine will be produced on industrial scale in a suitable expression host. Therefore, the peptide is optimized according to the E. coli, as it is the most suitable and affordable expression system for vaccine development. Hence, it is cloned in PET28a (+) vector for further expression analysis. After testing in vivo and in vitro, this vaccine may be considered as a future therapeutic.

This section has been added in the supporting information.

For the construction of potential vaccine, 95 M protein sequences of SARS-CoV-2 were retrieved from ViPR database. M protein is one of the structural proteins which is the utmost abundant constituent of the coronaviruses [19, 20] enabling it to conquer and assemble viral particles into the host cell. M protein with UniprotKB ID: P0DTC5 (VME1_SARS2) was found to be the most antigenic viral protein. The antigenicity estimation of viral protein was done using Vaxijen 2.0 online server [21] . For higher specificity, the threshold value was set at ≥ 0.4. The antigenicity for M protein was found to be 0.5102 by the full-length protein antigenicity analysis that indicated it as a probable antigen. Then, the protein was used for further investigation.

The calculation of physiochemical properties of SARS-CoV-2 M protein via ProtParam determined that it contains 222 amino acids and the molecular weight was found to be 25146.62

Da [22] . The theoretical isoelectric point (PI) was obtained as 9.51 that specifies it as positive in nature as isoelectric point above 7.0 shows positively charged proteins [23] . Protparam calculated instability-index (II) of 39.14; which categorized our protein as a stable one. Aliphatic index of 120.86 designates that protein is thermo-stable over a wide temperature assortment [24, 25] . Prediction of secondary structure via PSIPRED [26] and 3D structure via I-TASSER [27] showed that M protein exhibited 10% α helixes, 50% β sheets, and 40% loops ( Figure S2a ).

Moreover, the prediction of 6 disulfide bonds (S-S) and the allocation of scores in Table 1 was done via DiANNA1.1 tool [28] . Prediction of transmembrane topology was completed using an online tool TMHMM ( Figure S2b ). The residues from 1 to 19 and 74 to 77 were found to be surface exposed, while residues from 40 to 50 and 101 to 122 were found inside the transmembrane-region. Residues from 20 to 39 and 78 to 100 were found to be buried within the core-region of the M protein ( Figure 1 ). against distinctive viral infection. Here, we anticipated amino acid screened based methods for the analysis of potential B-cell epitopes. We used consensus-based approach to predict potential B-cell epitopes.

Total 6 linear epitopes were predicted by Bepipred Linear Epitope Prediction with threshold score of 0.350. The tool uses Hidden Markov model-based algorithm which is one of the best linear epitopes calculating methods. The minimum score for linear epitope prediction was 0.010 and the maximum score was 1.623. Moreover, the average prediction score was found to be -0.779. After analyzing all the data, we found that the peptide sequences from 206-215 amino acids can speed up the desired immune response and declared as B-cell epitopes. The results obtained from this method are explained in Figure S3a and Table 2 . Antigenicity of experimentally known amino acid epitopes was examined by the Kolaskar and Tongaonkar method [29] . The maximum tendency of antigenicity was found to be 1.235 and the minimum value was 0.904. The average observed value was 1.053. Threshold value was adjusted at 1.00 and all the values greater than 1.00 were candidates of antigenic factors. Total 7 epitopes were found satisfying the threshold value and selected for further analysis. They were found to have potential to initiate B-cell response. The results of antigenicity analysis are shown in Figure S3b and Table 3 . Good surface accessibility is an important factor for an effective B-cell epitope. Therefore, Emini surface accessibility tool was utilized for this prediction. The default threshold value was found as 1.000 and total 7 epitopes were selected passing the default value of threshold. The more accessible region was 88 to 94 amino acid residues and average observed value was 1.00.

Although the minimum value was found to be 0.078 and maximum value was 5.199 . The result are shown in Figure S3c and Table 4 . 

Beta turn is surface exposed and hydrophilic in nature. It plays a dynamic part in the commencement of immune system defense response. For the prediction of beta-turn in M protein, Chou and Fasman beta turn evaluating algorithm was used [30] . B-cell epitopes were further tested for conservancy analysis using IEDB conservancy analysis tool. Total 5 selected epitopes were used in conservancy analysis that were choosed to be utilized in vaccine construction. These epitopes were found to be conserved with maximum conservancy at 100% coverage and identity.

We selected homo sapiens as MHC source and the SMM method to investigate diverse set of MHC HLA alleles in humans. This tool provides an output interface for HLA-binding affinity of (Table S1 ).

630 conserved predicted epitopes with the IC50 less than 200 were found to interact with MHC Class-II alleles. Out of 630 epitopes, 38 were selected that were interacting with more than 6 MHC Cass-II alleles. 18 epitopes were finalized for further analyses because of allergencity, toxicity and antigenicity. The core epitopes IKLIFLWLL is considered to be the top binder as it followed by LYIIKLIFL and LFLTWICLL are supposed to bind with 10 and 9 alleles, respectively (Table S2) .

For multi-epitopic vaccine chimera construction, a total of 6 B-cell epitopes, 15 MHC Class-I epitopes and 18 MHC Class-II epitopes were used. 50S ribosomal protein L7/L12 with UniProt ID: P9WHE3 was used as an adjuvant in order to build the vaccine construct. The adjuvant was linked to the first B-cell epitope using an EAAAK linker at the amino (N) terminus to generate a specific immune response. Furthermore, the linkage of B-cell and MHC Class-I epitopes,

GPGPG linkers were utilized. MHC Class-II epitopes were connected using AAY linkers. The overlapping regions of B-cell epitopes, CTL and HLT repitopes were merged to reduce the size of vaccine (Table S3 ). For protein identification and purification process, 6x His tag was Figure   S4 and Figure S5 ). The IEDB conservancy analysis tool evaluated the conservancy of the predicted epitopes (Table S1 & Table S2 ).

The antigenicity of the vaccine protein along with adjuvant was calculated using VaxiJen 2.0 web server and it was predicted to be 0.5430. The antigenicity of vaccine construct without adjuvant was found to be 0.6604. It was concluded from the results that the vaccine construct is antigenic in nature either it is attached with adjuvant or not. The vaccine was found to be nonallergenic according to the results of AllerTOP v2 either the adjuvant is attached or not. The toxicity of the protein was found to be non-toxic by Toxinpred with or without adjuvant. indicates that the protein is not hydrophilic in nature [33] . Our vaccine construct showed higher solubility rate calculated through SOLpro server with the score 0.500000.

Extrapolation of secondary structure was performed using Raptor X tool that analyzed the actual nature of the protein. Results provided various aspects of the protein and it was concluded that the protein contains 48% helix, 9% beta strands and 41% coils. Total 46% protein content was found to be exposed, 25% was found to be medium exposed and 17% buried. There were total 15% residues found to be present in disordered domain.

Tertiary structure assessment of the protein 1 best tertiary structure model of chimeric vaccine construct were constructed by 3D PRO by Scratch Protein online server. On the basis of high coverage values, the models were predicted using top 10 threading templates. In this study, the model having highest coverage-score was selected for refinement procedures. [34] .

GalaxyRefine tool provided total five models of the vaccine chimera after refinement. Various parameters were considered in the process of refinement such as GDT-HA (0.8875), RMSD (0.568), and MolProbity (2.000). The calculation of clash score was found to be 17.8, score of poor rotamers was 1.3 and the Ramachandran score was predicted as 97.1%. For latter investigations, model 3 was selected as it was found to be the most authentic one.

The refined tertiary structure was validated by RAMPAGE server. The structure was analyzed, and Ramachandran plot was produced for the structure of protein ( Figure S6 ). There were total 92% region that was in favored region of the plot before refinement and only 8% structural region was found to be in allowed region. 0% structure was in the outlier region. After performing the refinement, better results were generated by RAMPAGE. According to these results, total 94.3% residues in favoured region, 5.3% residues in allowed region and 0.4% residues in outlier region.

Molecular docking was performed in order to estimate the interaction between refined vaccine model and ligand binding domain of immune receptor TLR3 by using online server Cluspro2.0

for protein-protein docking [35] . Docking gives multiple models for analysis. After analyzing all These values indicate stable vector expression in E. coli, as GC content ranges between 30-70% and is considered as optimum ( Figure S7a) . Finally, the optimized sequence was cloned in pET28 (+) vector to make a recombinant plasmid after being amplified through in-silico PCR by using SnapGene software (Figures S7b-c) .

Immune simulation was carried by C-ImmSim server. This exhibits immune response as that of real immune response. The primary response was marked by the increased level of IgM. High B- cell population characterized secondary and tertiary responses. As the concentration of antigen decreased; the concentration of IgM, IgG1+IgG2 and IgG+ IgM increased ( Figure S8a ). The results also showed the production of memory cell upon subsequent exposure ( Figure S8b ).

Along with this, there was also an increased concentration of helper (TH) and cytokines ( Figures   S8c-d) .

With the rise in global crises of this deadly virus, the need of the hour is to take a step forward in order to find the cure against this novel disase. With the advancement of computational aided sequence-based technology, bioinformatics tools provide a vital approach in peptide-based vaccine designing. The peptide based vaccine design in other viruses like dengue virus, chikungunya, rhino virus, and SLE virus have already proved a potential pipeline of viral targeting [36] . RNA based SARS-CoV-2 shows multiple resistance due to high mutation in its genome. Because of this reason, the outer protein or membrane protein (M Protein) covering the virus is the main focus of this study [37] . However, the physicochemical and secondary structure analysis of the targeted protein reveals it to be highly antigenic helical candidate to design vaccine. Moreover, the PDB structure of the related protein was also designed by using bioinformatics software due to its unavailability in different protein data banks. Overall, this study demonstrates the design of multi-epitope-based subunit peptide vaccine by means of insilico approaches to increase the both humoral and cell-mediated immune response.

In the past, B-cells were the only source for the design of a potential vaccine. However, with the innovation in computational biology, targeting major histocompatibility complex (MHC) T-cells with most interacting human leukocyte antigen (HLA) scheme is the new field of clinical research [38] . With interval, the antigenic drift may liberate the antigen from the antibody memorial response. Although, the T-cell immunity gives long-lasting immune response [39] , the journey of epitope to become vaccine involves some strict criteria. contribute in the epitope development [40] . However, three of them provide the peptide sequence of epitopes for further analysis. On the other hand, IEDB also comes in handy when analysis was made on binding and processing prediction of T cells [40] . Here, we screened out potential T-cell epitopes with IC50 value less than 200 making it highly active against its targeted allele.

Considering the consensus approach, epitopes interacting with more than five MHC Class-I and MHC Class-II get separated for further screening.

The epitopes get strengthen debate after proving best among different criteria. sequence is possibly allergenic if it has an at least six contiguous amino acids when compared to known allergens amino acid database [42] . Hence, our selected epitopes according to AllerTop v.2.0 did not accomplish the criteria for the FAO/WHO assessment scheme of allergenicity prediction and thus characterized as a nonallergen. The putative antigenic epitopes with no allergenicity and toxicity making immunoreactive peptides for our further studies [43] .

Conservancy of screened epitopes, among all the strains of HCoV, make them potential candidate towards vaccine development. IEDB screened out epitope shows good conservancy in fraction of protein sequences and identity is the degree of correspondence among strains.

Moreover, population coverage analysis for T-cell was done by IEDB as MHC molecules are enormously polymorphic and found in thousand diverse human MHC (HLA) alleles [44] . A study on 50S ribosomal protein L7/L12 shows that it is considered to be involved in increasing the response of vaccine in the pathogen recognition and immune system activation, therefore, selected as adjuvant to increase the immunoreactive property [45] . Initially selected B

and T-cell epitopes were fused using suitable linkers in order to develop the multiepitope subunit Next step in the vaccine construction is crucial to achieve promising impact. Immunoinformatics involving computational methods such as molecular docking has been recognized as a potent tool when it comes to predict the protein-protein interaction. Various studies have reported the involvement of TLR particularly TLR3 in generating immune response against SARS-CoV-2. A research has described the specificity of TLR3 in the action of innate immunity [47] . Docking is done by Clustpro2.0 showing that the model 9 of the vaccine-receptor complex exhibits promising interaction proximal to reference structure. Least energy value of the docked complex shows the stable interaction and less RMSD from the original conformation. The presence of strong electrostatic, van der Waals, hydrogen bonds and hydrophobic interaction making ligand in stable configuration inside the binding pocket of the receptor [37] . Docking gives us single snapshot of complex physiological movement. Therefore, there is a need to study protein-protein interaction in a more flexible environment [33] . For this purpose, molecular dynamics simulation was performed mimicking the natural behavior of our dynamic system. The results indicated various factors that are explained in Figure 4 . The figure explains the MNA mobility of the complex structure, deformability values, B-factor, eigenvalues, c-variance and elasticity of the complex. Based on maximum eigenvalue, the constructed complex shown to be stable and reveals less chances of deformation during immune response. Immune simulation of the designed construct was discovered by co-variance matrix analysis and its output was in accordance with the immune responses. The humoral response was anticipated to be produced as a result of vaccine introduction in the body [48, 49] .

Typical immune response was obtained by immune simulation. The immune response is enhanced as the antigen exposure is increased repeatedly. It was evident that memory B-cells were developed that are consistent for several months. There were also the development of memory T-cells along with helper T-cells simulation. There was an indication of increased level of IL-2 after first injection. It had been studied that the T-cell response is higher for structural protein [37] . In order to validate the designed vaccine, it was tested for immunoreactivity [50] , which was done by expressing it in E. coli [51] . For maximum expression the codon was optimized according to host which results in CAI of 0.96 and GC content of 51.8%.

However, many vaccine candidates have been evaluated using in-silico approaches that could be possible vaccine candidates but no vaccine has been efficiently designed using M-protein of the novel coronavirus. Furthermore, in previously designed vaccines, immune simulation and vaccine cloning were not performed. Some vaccine candidates have a low population coverage as compared to our designed vaccine chimera. Therefore, it would prove an efficient possible vaccine candidate if it is carried out in vitro and in vivo studies.

In this present study, computational approaches were used to develop an effective vaccine against SARS-CoV-2 infections. Providence of artificial environment favors the illustrated approach to design promising vaccine as a time saving and cost effective strategy due to minimal chances of failure. In silico vaccine with a good immune response and population coverage is the potential candidate for clinical trials. In silico immune simulation showed immune response in accordance with the clearing of antigen. Computational cloning in PET28a (+) plasmid using snapgene showed good protein expression. However, to ensure the efficacy of vaccine construct against COVID-19, experimental validation is essential. In many trials, peptide vaccines had shown good results with a better immune response, so this vaccine construct may be considered.

Coronavirus pathogenesis

Epidemiology, genetic recombination, and pathogenesis of coronaviruses

Coronavirus as a possible cause of severe acute respiratory syndrome

Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia

Real-time tentative assessment of the epidemiological characteristics of novel coronavirus infections in Wuhan

The epidemiology and pathogenesis of coronavirus disease (COVID-19) outbreak

for the Zhongnan Hospital of Wuhan University Novel Coronavirus Management and Research Team, Evidence-Based Medicine Chapter of China International Exchange and Promotive Association for Medical and Health Care (CPAM). A rapid advice guideline for the diagnosis and treatment of

Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin. bioRxiv

Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan, China

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An overview of viral structure and host response

The novel coronavirus 2019 (SARS-CoV-2) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells. bioRxiv

Molecular immune pathogenesis and diagnosis of COVID-19

Diagnosis of the Coronavirus disease (COVID-19): rRT-PCR or CT?

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (SARS-CoV-2) in vitro

The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro

Vaccine development against coronavirus (2003 to present): An overview, recent advances, current scenario, opportunities and challenges

Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid

Characterization of a coronavirus: I. Structural proteins: Effects of preparative conditions on the migration of protein in polyacrylamide gels

VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines

Protein identification and analysis tools on the ExPASy server

Protein extraction and precipitation

The proteomics protocols handbook

A simple method for displaying the hydropathic character of a protein

Scalable web services for the PSIPRED Protein Analysis Workbench

The I-TASSER Suite: protein structure and function prediction

DiANNA 1.1: an extension of the DiANNA web server for ternary cysteine classification

A semi-empirical method for prediction of antigenic determinants on protein antigens

Turns in peptides and proteins

Prediction of chain flexibility in proteins

Thermostability and aliphatic index of globular proteins

Exploring dengue genome to construct a multi-epitope based subunit vaccine by utilizing immunoinformatics approach to battle against dengue infection

Scoring function for automated assessment of protein structure template quality

PatchDock and SymmDock: servers for rigid and symmetric docking

Computational vaccinology based development of multi-epitope subunit vaccine for protection against the Norovirus infections

Coronavirus infections and immune responses

Strategies for vaccine design for corona virus using Immunoinformatics techniques

Metabolic and epigenetic coordination of T cell and macrophage immunity

Design of an epitope-based peptide vaccine against spike protein of human coronavirus: an in silico approach. Drug Design

Immunoinformatics and structural vaccinology driven prediction of multi-epitope vaccine against Mayaro virus and validation through in-silico expression

Value of eight-amino-acid matches in predicting the allergenicity status of proteins: an empirical bioinformatic investigation

In silico predicted mycobacterial epitope elicits in vitro T-cell responses

Human coronavirus NL63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in Hong Kong, China

Recombinant L7/L12 ribosomal protein and gamma-irradiated Brucella abortus induce a T-helper 1 subset response from murine CD4+ T cells

Designing a multi-epitopic vaccine against the enterotoxigenic Bacteroides fragilis based on immunoinformatics approach

Toll-like receptor 4: A promising crossroads in the diagnosis and treatment of several pathologies

In-silico design of a multi-epitope vaccine candidate against onchocerciasis and related filarial diseases

Excavating SARScoronavirus 2 genome for epitope-based subunit vaccine synthesis using immunoinformatics approach

Peptides for immunological purposes: design, strategies and applications

Recombinant protein expression in Escherichia coli: advances and challenges

We acknowledge the support of Department of Biotechnology, University of Central Punjab, Lahore. The authors would like to thank the University of Nizwa for the generous support of this project. We thank analytical and technical staff for assistance.J o u r n a l P r e -p r o o f