Multi-epitope Construct Research Articles

B1 protein: a novel cell penetrating protein for in vitro and in vivo delivery of HIV-1 multi-epitope DNA constructs.

ObjectivesEnhancement of the potential ability of biomacromolecules to cross cell membranes is a critical step for development of effective therapeutic vaccine especially DNA vaccine against human immunodeficiency virus-1 (HIV-1) infection. The supercharged proteins were known as powerful weapons for delivery of different types of cargoes such as DNA and protein. Hence, we applied B1 protein with + 43 net charges obtained from a single frameshift in the gene encoding enhanced green fluorescent protein (eGFP) for delivery of two multi-epitope DNA constructs (nef-vpu-gp160-p24 and nef-vif-gp160-p24) in vitro and in vivo for the first time. For this purpose, B1 protein was generated in bacterial expression system under native conditions, and used to interact with both DNA constructs.ResultsOur data indicated that B1 protein (~ 27 kDa) was able to form a stable nanoparticle (~ 80–110 nm) with both DNA constructs at nitrogen: phosphate (N: P) ratio of 1:1. Moreover, the transfection efficiency of B1 protein for DNA delivery into HEK-293T cell line indicated that the cellular uptake of nef-vif-gp160-p24 DNA/ B1 and nef-vpu-gp160-p24 DNA/ B1 nanoparticles was about 32–35% with lower intensity as compared to TurboFect commercial reagent. On the other hand, immunization of BALB/c mice with different modalities demonstrated that B1 protein could enhance the levels of antibody, IFN-gamma and Granzyme B eliciting potent and strong Th1-directed cellular immunity.ConclusionGenerally, our findings showed the potency of B1 protein as a promising gene delivery system to improve an effective therapeutic vaccine against HIV-1 infection.

Development of multiepitope therapeutic vaccines against the most prevalent high-risk human papillomaviruses.

Aim: Our goal was the development of DNA- or peptide-based multiepitope vaccines targeting HPV E7, E6 and E5 oncoproteins in tumor mouse model. Materials &methods: After designing the multiepitope E7, E6 and E5 constructs from four types of high risk HPVs (16, 18, 31 & 45) using bioinformatics tools, mice vaccination was performed by different homologous and heterologous modalities in a prophylactic setting. Then, anti-tumor effects of the best prophylactic strategies were studied in a therapeutic setting. Results: In both prophylactic and therapeutic experiments, groups receiving homologous E7+E6+E5 polypeptide, and heterologous E7+E6+E5 DNA prime/polypeptide boost were successful in complete rejection of tumors. Conclusion: The designed multiepitope constructs can be considered as promising candidates to develop effective therapeutic HPV vaccines.

Comparative analysis of two HIV-1 multiepitope polypeptides for stimulation of immune responses in BALB/c mice

A licensed vaccine against human immunodeficiency virus-1 (HIV-1) infection has not become available up to now. Hence, it is more rational to use immune-informatics tools for prediction of T cell epitopes (in silico study) and development of an effective epitope-driven vaccine against hypervariable pathogens. Multiepitope vaccines were considered as the next generation of an effective vaccine against HIV-1 infection. In the current study, we developed two different constructs encoding T cell epitopes derived from Nef, Vif, Vpu, Gp160 and P24 proteins in BALB/c mice. To overcome their poor immunogenicity, four different cell penetrating peptides (MPG and HR9 for DNA delivery, and CyLoP-1 and LDP-NLS for protein delivery), Montanide adjuvant, and heterologous prime-boost immunization strategy were utilized. The generation of cytokines, Granzyme B, and total IgG and its subclasses was determined using ELISA. Our data indicated that the levels of IFN-γ and Granzyme B in mice injected with Nef-Vif-Gp160-P24 multiepitope constructs were higher than those immunized with Nef-Vpu-Gp160-P24 multiepitope constructs. Moreover, the heterologous DNA priming/ multiepitope peptide boosting in both Nef-Vif-Gp160-P24 and Nef-Vpu-Gp160-P24 regimens induced significantly high antigen-specific IgG2a and IgG2b responses in comparison with other groups. There was no significant difference between MPG and HR9 as well as CyLoP-1 and LDP-NLS as a delivery system for enhancement of immune responses. Generally, the heterologous DNA prime/ multiepitope peptide boost modalities for both constructs could significantly enhance the levels of IgG2a, IgG2b, IFN-γ, and Granzyme B directed toward Th1 immune responses as compared to homologous prime/ boost with DNA or polypeptide constructs.

Novel Chimeric Multiepitope Vaccine for Streptococcosis Disease in Nile Tilapia (Oreochromis niloticus Linn.)

Streptococcus agalactiae is a causative agent of streptococcosis disease in various fish species, including Nile tilapia (Oreochromis niloticus Linn.). Vaccination is an effective disease prevention and control method, but limitations remain for protecting against catastrophic mortality of fish infected with different strains of streptococci. Immunoproteomics analysis of S. agalactiae was used to identify antigenic proteins and construct a chimeric multiepitope vaccine. Epitopes from five antigenic proteins were shuffled in five helices of a flavodoxin backbone, and in silico analysis predicted a suitable RNA and protein structure for protein expression. 45F2 and 42E2 were identified as the best candidates for a chimeric multiepitope vaccine. Recombinant plasmids were constructed to produce a recombinant protein vaccine and DNA vaccine system. Overexpressed proteins were determined to be 30 kDa and 25 kDa in the E. coli and TK1 systems, respectively. The efficacy of the chimeric multiepitope construct as a recombinant protein vaccine and DNA vaccine was evaluated in Nile tilapia, followed by S. agalactiae challenge at 1 × 107 CFU/mL. Relative percentage survival (RPS) and cumulative mortality were recorded at approximately 57–76% and 17–30%, respectively. These chimeric multiepitope vaccines should be applied in streptococcosis disease control and developed into a multivalent vaccine to control multiple diseases.

Design and expression of polytopic construct of cathepsin-L1, SAP-2 and FhTP16.5 proteins of Fasciola hepatica.

The enzyme-linked immunosorbent assay (ELISA) technique can play an important role in the early detection of fascioliasis. However, they have some diagnostic limitations, including cross-reaction with other helminths. It seems that the combination of recombinant parasite proteins as antigen can reduce these problems. Hence, the present study was aimed to design and confirm the antigenic recombinant multi-epitope (rMEP) construct of three protein epitopes (linear and conformational B-cell epitopes) of the parasite using immunoinformatic tools. For this purpose, the tertiary structures of Fasciola hepatica cathepsin-L1, saposin-like protein 2 and 16.5-kDa tegument-associated protein were predicted using the I-TASSER server. Validation of the modelled structures was performed by Ramachandran plots. The antigenic epitopes of the proteins were achieved by analysing the features of the IEDB server. The synthesized gene was cloned into the pET-22b (+) expression vector and transformed into the Escherichia coli BL21. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to verify and analyse the expression of the rMEP protein. Western blotting was utilized to confirm rMEP protein immunogenicity in two forms, one using an anti-His tag antibody and the other with human pooled sera samples (fascioliasis, non-fascioliasis and negative control sera). Our results demonstrated that the rMEP designed for the three proteins of F. hepatica was highly antigenic, and immune-detection techniques confirmed the antigen specificity. In conclusion, the presented antigenic multi-epitope may be very helpful to develop serodiagnostic kits such as indirect ELISA to evaluate the proper diagnosis of fascioliasis in humans and ruminants.

Potential druggable proteins and chimeric vaccine construct prioritization against Brucella melitensis from species core genome data

The Brucella melitensis chronic infection and drug resistance emerged as a severe health problem in humans and domestic cattle. The pathogens fast genome sequences availability fetched the possibility to address novel therapeutics targets in a rationale way. We acquired the core genes set from 56 B. melitensis publically available complete genome sequences. A stringent bioinformatics layout of comparative genomics and reverse vaccinology was followed to identify potential druggable proteins and multi-epitope vaccine constructs from core genes. The 23 proteins were shortlisted as novel druggable targets based on their role in pathogen-specific metabolic pathways, non-homologous to human and human gut microbiome proteins and their druggability potential. Furthermore, potential chimeric vaccine constructs were generated from lead T and B-cell overlapped epitopes in combination with immune enhancer adjuvants and linkers sequences. The molecular docking and MD simulation analyses ensured stable molecular interaction of a finally prioritized vaccine construct with human immune cells receptors.

In silico screening of antigenic B-cell derived T-cell epitopes and designing of a multi-epitope peptide vaccine for Acinetobacter nosocomialis

Globally, antibiotic-resistant and tolerated bacterial isolates of Acinetobacter species are imposing high financial cost on health care systems and as such, molecular targets with promising immune protection could provide substantive benefits to human healthcare. Here, we performed an in silico based proteome-wide screening for antigenic B-cell derived T-cell epitopes and their following use to design a multi-epitope peptide vaccine that can effectively engage the host immune system against Acinetobacter nosocomialis SSA3 strain. Epitopes of the fimbrial biogenesis outer membrane usher FimD protein: YQQGINNYL and YRTNYTTVG were revealed appropriate for multi-epitope peptide construct designing. This protein has no homology to the host, essential to the pathogen survival and is localized at the pathogen surface. The predicted epitopes have high affinity for the highly expressed DRB*0101 allele in humans based on the lowest IC50 value in MHCPred and have an exo-membrane topology for efficient immune system recognition. The designed multi-epitope peptide vaccine is composed of the mentioned shortlisted antigenic epitopes linked to each other through a GPGPG linker, and an EAAAK linker that joined the multi-epitope peptide to the Cholera B subunit from Vibrio cholera as an adjuvant to increase vaccine construct antigenicity. The vaccine construct was docked and simulated with a transmembrane toll-like receptor (TLR4) that revealed construct stable binding with the TLR4 through the adjuvant, allowing the epitopes exposed to the host immune system essential for generating effective innate and long-lasting adaptive immunity. The designed multi-epitope peptide vaccine may prompt the development of a vaccine to control refractory and deleterious A. nosocomialis infections.

Design and in vitro delivery of HIV-1 multi-epitope DNA and peptide constructs using novel cell-penetrating peptides.

Developing an effective HIV vaccine that stimulates the humoral and cellular immune responses is still challenging because of the diversity of HIV-1 virus, polymorphism of human HLA and lack of a suitable delivery system. Using bioinformatics tools, we designed a DNA construct encoding multiple epitopes. These epitopes were highly conserved within prevalent HIV-1 subtypes and interacted with prevalent class I and II HLAs in Iran and the world. The designed DNA construct included Nef60-84, Nef126-144, Vpr34-47, Vpr60-75, Gp16030-53, Gp160308-323 and P248-151 epitopes (i.e., nef-vpr-gp160-p24 DNA) which was cloned into pET-24a(+) and pEGFP-N1 vectors. The recombinant polyepitope peptide (rNef-Vpr-Gp160-P24; ~ 32kDa) was successfully generated in E. coli expression system. The pEGFP-nef-vpr-gp160-p24 and rNef-Vpr-Gp160-P24 polyepitope peptide were delivered into HEK-293T cells using cell-penetrating peptides (CPPs). The MPG and HR9 CPPs, as well as the novel LDP-NLS and CyLoP-1 CPPs, were utilized for DNA and peptide delivery into the cells, respectively. SEM results confirmed the formation of stable MPG/pEGFP-N1-nef-vpr-gp160-p24, HR9/pEGFP-N1-nef-vpr-gp160-p24, LDP-NLS/rNef-Vpr-Gp160-P24 and CyLoP-1/rNef-Vpr-Gp160-P24 nanoparticles with a diameter of < 200nm through non-covalent bonds. MTT assay results indicated that these nanoparticles did not have any major toxicity in vitro. Fluorescence microscopy, flow cytometry and western blot data demonstrated that these CPPs could significantly deliver the DNA and peptide constructs into HEK-293T cells. The use of these CPPs can be considered as an approach in HIV vaccine development for in vitro and in vivo delivery of DNA and peptide constructs into mammalian cells.

Development a multi-epitope driven subunit vaccine for immune response reinforcement against Serogroup B of Neisseria meningitidis using comprehensive immunoinformatics approaches

Serogroup B of Neisseria meningitidis is the main cause of mortality due to meningococcal meningitis. Despite of many investigations, there is still no effective vaccine to prevent this serious infection. Therefore, this study was conducted to design a multi-epitope based vaccine through immunoinformatics approaches. The T CD4+ and TCD8+ cells along with IFN-γ inducing epitopes were selected from TspA, FHbp, NspA, TbpB, PilQ and NspA antigens form serogroup B of Neisseria meningitidis. Furthermore, to induce strong helper T lymphocytes (HTLs) responses, Pan HLA DR-binding epitope (PADRE) was used. In addition, loop 5 and 7 of the PorB as a TLR2 agonist were added to the vaccine construct. Physico-chemical properties, secondary and tertiary structures of the proposed construct were assessed. Finally, homology modeling, refinement and molecular docking were carried out to evaluated the construct tertiary structure and protein-protein interaction, respectively. By fusing the CTL, HTL and IFN-γ predicted epitopes along with suitable adjuvant and linkers, a multi-epitope vaccine was constructed with a TAT sequence of HIV at the N-terminal. Immunoinformatics analyses confirmed a soluble and non-allergic protein with a molecular weight of 62.5 kDa and high antigenicity. Furthermore, the stability of the multi–epitope construct was established and showed strong potential to generate humoral and cell-mediated immune responses. In addition, through molecular docking and dynamic simulation, the microscopic interaction between the vaccine construct and TLR-2 were verified. In summary, immunoinformatics analysis demonstrated that the constructed multi-epitope vaccine had a strong potential of T and B-cell stimulation and it could possibly be used for prophylactic or therapeutic aims to protect against serogroup B of N. meningitidis.

Specific immune responses induced by multi-epitope DNA derived from Mycobacterium tuberculosis DosR antigens.

One third of the world population are latently infected with Mycobacterium tuberculosis and are at the risk of reactivation of tuberculosis (TB). The most effective strategy for control of TB worldwide is the development of a vaccine that inhibits progression of latent TB to active infection. In this study, two optimized constructs consisting of multi-epitopes DNA derived from three latency antigens Rv2029c, Rv2031c, and Rv2627c fused with or without light chain 3 (LC3) are synthetized. The immunogenicity effectiveness of two DNA constructs was evaluated in the mouse model. LC3-fused multi-epitope DNA construct induced strong specific Th1 immune responses with high increase in IFN-γ+ CD4+ and IL-2+ CD4+ T cell populations (both with p < 0.0001) and IFN-γ+ IL-2+ CD4+ T cell population (p < 0.0001) compared with empty vector, BCG, and multi-epitope DNA construct groups. The LC3-fused construct induced IFN-γ+ CD8+ T cell population (p < 0.0001) compared with empty vector and BCG groups but could not induce the T cell population compared with construct without LC3. Importantly, LC3-fused DNA construct did not induce epitope-specific IL-4 and IL-10 from CD4+ and CD8+ T cell populations. The results indicated that LC3-fused multi-epitope DNA construct has a potential to be investigated for future development of a new TB vaccine.

An immunoinformatics-derived DNA vaccine encoding human class II T cell epitopes of Ebola virus, Sudan virus, and Venezuelan equine encephalitis virus is immunogenic in HLA transgenic mice

ABSTRACTImmunoinformatics tools were used to predict human leukocyte antigen (HLA) class II-restricted T cell epitopes within the envelope glycoproteins and nucleocapsid proteins of Ebola virus (EBOV) and Sudan virus (SUDV) and the structural proteins of Venezuelan equine encephalitis virus (VEEV). Selected epitopes were tested for binding to soluble HLA molecules representing 5 class II alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, and DRB1*1501). All but one of the 25 tested peptides bound to at least one of the DRB1 alleles, and 4 of the peptides bound at least moderately or weakly to all 5 DRB1 alleles. Additional algorithms were used to design a single “string-of-beads” expression construct with 44 selected epitopes arranged to avoid creation of spurious junctional epitopes. Seventeen of these 44 predicted epitopes were conserved between the major histocompatibility complex (MHC) of humans and mice, allowing initial testing in mice. BALB/c mice vaccinated with the multi-epitope construct developed statistically significant cellular immune responses to EBOV, SUDV, and VEEV peptides as measured by interferon (IFN)-γ ELISpot assays. Significant levels of antibodies to VEEV, but not EBOV, were also detected in vaccinated BALB/c mice. To assess immunogenicity in the context of a human MHC, HLA-DR3 transgenic mice were vaccinated with the multi-epitope construct and boosted with a mixture of the 25 peptides used in the binding assays. The vaccinated HLA-DR3 mice developed significant cellular immune responses to 4 of the 25 (16%) tested individual class II peptides as measured by IFN-γ ELISpot assays. In addition, these mice developed antibodies against EBOV and VEEV as measured by ELISA. While a low but significant level of protection was observed in vaccinated transgenic mice after aerosol exposure to VEEV, no protection was observed after intraperitoneal challenge with mouse-adapted EBOV. These studies provide proof of concept for the use of an informatics approach to design a multi-agent, multi-epitope immunogen and provide a basis for further testing aimed at focusing immune responses toward desired protective T cell epitopes.

Designing a Novel Multi-epitope DNA- Based Vaccine Against Tuberculosis: In Silico Approach

Background: Tuberculosis remains a major global threat. Two billion of the world’s population is latently infected with Mycobacterium tuberculosis and is at the risk of progression to active disease. Bacillus Calmette-Guerin (BCG), as the only licensed vaccine, has prophylaxis strategy, which protects children from disseminated form of tuberculosis. Therefore, postexposure vaccine strategy, which targets individuals with latent tuberculosis infection, is an important strategy to control this disease globally. Objectives: In the present study, we designed a novel postexposure multi-epitope DNA construct based on 3 latency-associated antigens of Rv2029c, Rv2031c, and Rv2627c and microtubule-associated protein light chain 3 (LC3) as a hallmark protein of the autophagy system. Methods: A mouse construct was designed based on predicted MHC class I-and class II-restricted T- cell epitopes that fused together tandemly. MHC class I-and class II-restricted epitopes were linked by AAY and GPGPG motifs, respectively. LC3 directly fused to the MHC class II-restricted epitopes at the C-terminus of the peptide. The varieties of expressed construct features were analyzed by bio- informatics tools. Finally, construct codons were optimized and mRNA structure of optimized construct was analyzed. Results: MHC class I-and class II-predicted epitopes showed a high potential to binding to human HLAs alleles, with global broad-spectrum population coverage. The construct had no allergenicity, and the analysis indicated a desirable antigenicity of the construct. The construct had several posttranslational modifications, no signal peptide, and cytoplasmic localization with high score. Also, mRNA analysis showed low ∆G which demonstrated high stability and efficient translation. Conclusions: The results revealed that the novel multi-epitope DNA construct could be an effective candidate in tuberculosis vaccine development, and it is qualified to investigate its potential to induce CD4 and CD8 T- cell immune response in the experimental animal model.

In silico analyses of heat shock protein 60 and calreticulin to designing a novel vaccine shifting immune response toward T helper 2 in atherosclerosis

Recent experiments demonstrated that atherosclerosis is a Th1 dominant autoimmune condition, whereas Th2 cells are rarely detected within the atherosclerotic lesions. Several studies have indicated that Th2 type cytokines could be effective in the reduction and stabilization of atherosclerotic plaque. Therefore, the modulation of the adaptive immune response by shifting immune responses toward Th2 cells by a novel vaccine could represent a promising approach to prevent from progression and thromboembolic events in coronary artery disease. In the present study, an in silico approach was applied to design a novel multi-epitope vaccine to elicit a desirable immune response against atherosclerosis. Six novel IL-4 inducing epitopes were selected from HSP60 and calreticulin proteins. To enhance epitope presentation, IL-4 inducing epitopes were linked together by AAY and HEYGAEALERAG linkers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Moreover, cholera toxin B (CTB) was employed as an adjuvant. A multi-epitope construct was designed based on predicted epitopes which was 320 residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this chimeric protein were analyzed using bioinformatics tools and servers. Based on bioinformatics analysis, a soluble, and non-allergic protein with 35.405kDa molecular weight was designed. Expasy ProtParam classified this chimeric protein as a stable protein. In addition, predicted epitopes in the chimeric vaccine indicated strong potential to induce B-cell mediated immune response and shift immune responses toward protective Th2 immune response. Various in silico analyses indicate that this vaccine is a qualified candidate for improvement of atherosclerosis by inducing immune responses toward T helper 2.

A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus (Coral Snake) Venom.

BackgroundEnvenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage.Methods and FindingsIn this work we describe the mapping, by the SPOT-synthesis technique, of potential B-cell epitopes from five putative toxins from M. corallinus, which were used to design two multiepitope DNA strings for the genetic immunisation of female BALB/c mice. Results demonstrate that sera obtained from animals that were genetically immunised with these multiepitope constructs, followed by booster doses of recombinant proteins lead to a 60% survival in a lethal dose neutralisation assay.ConclusionHere we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity.

A new strategy for development of hepatitis E vaccine: Epitope-based vaccines

ABSTRACT Vaccination is generally known as the most effective strategy for controlling infectious diseases. However, the traditional vaccine approaches are incapable of preventing emerging infectious diseases as well as antigenically hypervariable pathogens that are still a main threat for world health. Recent advances in computational approaches developed for epitope identification and in silico analysis have paved the way for the design of epitope-based vaccines against these kinds of pathogens. The aim of these novel vaccines is to induce immune responses in a more effective way than the conventional vaccines. In this review, we describe the principles of epitope-based vaccine design, along with the application of these approaches in design and development of high density multiepitope construct as the first epitope-based vaccine candidate against hepatitis E virus. Keywords: Epitope-based vaccine; Multiepitope construct; High epitope density; In silico vaccine design; Epitope selection; Optimization; Hepatitis E virus; Immunity

Chimeric Epitope Vaccine from Multistage Antigens for Lymphatic Filariasis.

Lymphatic filariasis, a mosquito-borne parasitic disease, affects more than 120 million people worldwide. Vaccination for filariasis by targeting different stages of the parasite will be a boon to the existing MDA efforts of WHO which required repeated administration of the drug to reduce the infection level and sustained transmission. Onset of a filaria-specific immune response achieved through antigen vaccines can act synergistically with these drugs to enhance the parasite killing. Multi-epitope vaccine approach has been proved to be successful against several parasitic diseases as it overcomes the limitations associated with the whole antigen vaccines. Earlier results from our group suggested the protective efficacy of multi-epitope vaccine comprising two immunodominant epitopes from Brugia malayi antioxidant thioredoxin (TRX), several epitopes from transglutaminase (TGA) and abundant larval transcript-2 (ALT-2). In this study, the prophylactic efficacy of the filarial epitope protein (FEP), a chimera of selective epitopes identified from our earlier study, was tested in a murine model (jird) of filariasis with L3 larvae. FEP conferred a significantly (P < 0.0001) high protection (69.5%) over the control in jirds. We also observed that the multi-epitope recombinant construct (FEP) induces multiple types of protective immune responses, thus ensuring the successful elimination of the parasite; this poses FEP as a potential vaccine candidate.

In silico analyses of Wilms׳ tumor protein to designing a novel multi-epitope DNA vaccine against cancer

Predefined and pre-weighted objective criteria and essential role of Wilms׳ tumor wild type gene (WT1) for maintaining transformed features of cancer cells confirm the high potency of WT1 as a valuable cancer antigen. The antigen was at the top of the ranking among 75 representative cancer antigens. In the present study, an in silico approach was launched to characterized novel CTL epitopes and design a novel multi-epitope DNA vaccine to elicit a desirable immune response against cancers over expressing WT1. Forty-four novel epitopes were described. A multi-epitope construct was designed based on predicted epitopes which is 310 residues in length. The vaccine candidate designed here displays acceptable population coverage (>65%) in different ethnicities as well as high probability of eliciting WT1 antibodies which both are pertinent goals in the context of appropriate multi-epitope vaccines. Various in silico analyses indicate that final vaccine is a qualified immunotherapy candidate capable of eliciting both CD4+ and CD8+ T cell responses.

Design and production of a multiepitope construct derived from hepatitis E virus capsid protein.

The aim of this study was to design a high density multiepitope protein, which can be a promising multiepitope vaccine candidate against Hepatitis E virus (HEV). Initially, conserved and antigenic helper T‐lymphocyte (HTL) epitopes in the HEV capsid protein were predicted by in silico analysis. Subsequently, a multiepitope comprising four HTL epitopes with high‐affinity binding to the HLA molecules was designed, and repeated four times as high density multiepitope construct. This construct was synthesized and cloned into pET‐30a (+) vector. Then, it was transformed and expressed in Escherichia coli BL21 cells. The high density multiepitope protein was purified by Ni‐NTA agarose and concentrated using Amicon filters. Finally, the immunological properties of this high density multiepitope protein were evaluated in vitro. The results showed that the high density multiepitope construct was successfully expressed and purified. SDS‐PAGE and Western blot analyses showed the presence of a high density multiepitope protein band of approximately 33 kDa. Approximately 1 mg of the purified protein was obtained from each liter of the culture media. Moreover, the purified multiepitope protein was capable of induction of proliferation responses, IFN‐γ ELISPOT responses and IFN‐γ and IL‐12 cytokines production in a significant level in peripheral blood mononuclear cells (PBMCs) isolated from HEV‐recovered individuals compared to the control group. In conclusion, the newly produced multiepitope protein can induce significant T helper type 1 responses in vitro, and can be considered as a novel strategy for the development of HEV vaccines in the future. J. Med. Virol. 87:1225–1234, 2015. © 2015 Wiley Periodicals, Inc.

Design and characterization of a chimeric multiepitope construct containing CfaB, heat‐stable toxoid, CssA, CssB, and heat‐labile toxin subunit B of enterotoxigenic Escherichia coli: a bioinformatic approach

Enterotoxigenic Escherichia coli (ETEC) strains are the most common cause of bacterial diarrhea in children in developing countries and travelers to these areas. Enterotoxins and colonization factors (CFs) are two key virulence factors in ETEC pathogenesis, and the heterogeneity of the CFs is the bottleneck in reaching an effective vaccine. In this study, a candidate subunit vaccine, which is composed of CfaB, CssA and CssB, structural subunits of colonization factor antigen I and CS6 CFs, labile toxin subunit B, and the binding subunit of heat-labile and heat-stable toxoid, was designed to provide broad-spectrum protection against ETEC. The different features of chimeric gene, its mRNA stability, and chimeric protein properties were analyzed by using bioinformatic tools. The optimized chimeric gene was chemically synthesized and expressed successfully in a prokaryotic host. The purified protein was used for assessment of bioinformatic data by experimental methods.

Designing and production of a chimeric multi-epitope construct of CfaB, ST toxoid, CssA, CssB and LTB against enterotoxigenic E. coli

Designing and production of a chimeric multi-epitope construct of CfaB, ST toxoid, CssA, CssB and LTB against enterotoxigenic E. coli