KRAS G12A somatic point mutation in adenocarcinomas is categorized clinically as ineligibility criteria for anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies. In this study, a modified G12A-K-ras epitope (139A) with sequence-specific modifications to improve immunogenicity was developed as a potential vaccine against G12A-mutant KRAS cancers. Additionally, coupling of the 139A epitope with a tetanus toxoid (TTD) universal T-cell epitope to improve antigenicity was also reported. To facilitate convenient oral administration, Lactococcus lactis, which possesses innate immunomodulatory properties, was chosen as a live gastrointestinal delivery vehicle. Recombinant L. lactis strains secreting a G12A mutated K-ras control and 139A with and without TTD fusion were generated for comparative immunogenicity assessment. BALB/c mice were immunized orally, and high survivability of L. lactis passage through the gastrointestinal tract was observed. Elevations in B-cell count with a concomitant titre of antigen-specific IgG and interferon-γ secreting T-cells were observed in the 139A treated mice group. Interestingly, an even higher antigen-specific IgA response and interferon-γ secreting T-cell counts were observed in 139A-TTD mice group upon re-stimulation with the G12A mutated K-ras antigen. Collectively, these results indicated that an antigen-specific immune response was successfully stimulated by 139A-TTD vaccine, and a TTD fusion was successful in further enhancing the immune responses.
Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing the malarial epitopes F2R(II)EBA and (NANP)3 as well as two T cell epitopes of the M. tuberculosis ESAT-6 antigen, generated in favour of mycobacterium codon usage elicited specific immune response against these epitopes. Immunised Balb/c mice demonstrated an increase in almost all of the IgG subclasses against both malarial epitopes and enhanced splenocyte proliferative response against the malarial epitopes as well as selected peptides of ESAT-6. Furthermore, flow cytometric analyses showed elevated numbers of CD4+ lymphocytes expressing IFN-gamma and IL-2 against the ESAT-6 peptides, suggesting a specific Th1-mediated response. This study demonstrated that expressing malarial and TB epitopes in a single rBCG construct induced appropriate humoral and cellular immune response against immunogenic epitopes from both organisms.
The most important targets for vaccine development are the proteins that are highly expressed by the microorganisms during infection in-vivo. A number of Mycobacterium tuberculosis (Mtb) proteins are also reported to be expressed in-vivo at different phases of infection. In the present study, we analyzed multiple published databases of gene expression profiles of Mtb in-vivo at different phases of infection in animals and humans and selected 38 proteins that are highly expressed in the active, latent and reactivation phases. We predicted T- and B-cell epitopes from the selected proteins using HLAPred for T-cell epitope prediction and BCEPred combined with ABCPred for B-cell epitope prediction. For each selected proteins, regions containing both T- and B-cell epitopes were identified which might be considered as important candidates for vaccine design against tuberculosis.
This study is focused towards developing a global consensus sequence of nonstructural protein 2 (NSP2), a protease of Chikungunya Virus (CHIKV) and predict immunogenic promiscuous T-cell epitopes based on various bioinformatics tools. To date, no epitope data is available for the Chikungunya virus in the IEDB database. In this study, 100 available nucleotide sequences of NSP2-CHIKV belonging to different strains were downloaded from the National Centre for Biotechnology Information (NCBI) database. The nucleotide sequences were subjected to translated sequencing using the EXPASY tool followed by protein alignment using the CLC workbench and a global consensus sequence for the respective protein was developed. IEDB tool was used to predict HLA-I and HLA-II binding promiscuous epitopes from the consensus sequence of NSP2-CHIKV. Thirty-four B-cell based epitopes are predicted and the promiscuous epitope is VVDTTGSTKPDPGD at position 341-354. Twenty-six MHC-I short peptide epitopes are predicted to bind with HLA-A. The promiscuous epitopes predicted to bind with HLA-A*01:01 are VTAIVSSLHY, SLSESATMVY, FSKPLVYY, QPTDHVVGEY at positions 317-326, 84-93, 535-544 and 15-24 with percentile ranks 0.17, 0.39, 0.51 and 0.81, respectively. Twenty-four MHC-II short peptide epitopes are predicted for HLA-DRB. The promiscuous epitope predicted to bind with HLA-DRB*01:01 is VVGEYLVLSPQTVLRS from 20-35 with a lowest percentile rank of 0.01. These predicted epitopes can be effective targets towards development of vaccine against CHIKV. Epitopes predicted in this study displayed good binding affinity, antigenicity and promiscuity for the HLA classes. These predicted epitopes can prove to be translationally important towards the development of CHIKV.
Mycobacterium bovis bacille Calmette-Guèrin (BCG) represents one of the most promising live vectors for the delivery of foreign antigens to the immune system. A recombinant BCG containing a synthetic gene coding for the malarial epitopes namely, the fragment 2 of region II of EBA-175 (F2R(II)EBA) and the repeat sequence of the circumsporozoite protein NANP generated in favour of mycobacterium codon usage using assembly PCR was constructed. Two T-cell epitopes of the 6-kDa M. tuberculosis early-secreted antigenic target (ESAT-6) antigen were also clone in the same construct. Expression of the synthetic gene was driven by the heat shock protein 65 (hsp65) promoter from M. tuberculosis and the signal peptide from the MPT63 antigen of M. tuberculosis. Expression of the composite epitopes was detected by Western blotting of the cell extract and culture supernatant of the recombinant clones using a specific rabbit polyclonal antibody against F2R(II)EBA. This study demonstrates the possibility of cloning and expressing immunogenic epitopes from causative agents of two important diseases: malaria and tuberculosis (TB) in a single recombinant BCG construct.
Approximate 70% of cobra venom is composed of cytotoxin (CTX), which is responsible for the dermonecrotic symptoms of cobra envenomation. However, CTX is generally low in immunogenicity, and the antivenom is ineffective in attenuating its in vivo toxicity. Furthermore, little is known about its epitope properties for empirical antivenom therapy. This study aimed to determine the epitope sequences of CTX using the immunoinformatic analyses and epitope-omics profiling. A conserved CTX was used in this study to determine its T-cell and B-cell epitope sequences using immunoinformatic tools and molecular docking simulation with different Human Leukocyte Antigens (HLAs). The potential T-cell and B-cell epitopes were 'KLVPLFY,' 'CPAGKNLCY,' 'MFMVSTPTK,' and 'DVCPKNSLL.' Molecular docking simulations disclosed that the HLA-B62 supertype exhibited the greatest binding affinity towards cobra venom cytotoxin. The namely L7, G18, K19, N20, M25, K33, V43, C44, K46, N47, and S48 of CTX exhibited prominent intermolecular interactions with HLA-B62. The multi-enzymatic-limited-digestion/liquid chromatography-mass spectrometry (MELD/LC-MS) also revealed three potential epitope sequences as 'LVPLFYK,' 'MFMVS,' and 'TVPVKR'. From different epitope mapping approaches, we concluded four potential epitope sites of CTX as 'KLVPLFYK', 'AGKNL', 'MFMVSTPKVPV' and 'DVCPKNSLL'. Site-directed mutagenesis of these epitopes confirmed their locations at the functional loops of CTX. These epitope sequences are crucial to CTX's structural folding and cytotoxicity. The results concluded the epitopes that resided within the functional loops constituted potential targets to fabricate synthetic epitopes for CTX-targeted antivenom production.
Enterovirus A71 (EV-A71) from the Picornaviridae family is an important emerging pathogen causing hand, foot, and mouth disease (HFMD) outbreaks worldwide. EV-A71 also caused fatal neurological complications in young children especially in Asia. On the basis of seroepidemiological studies from many Asian countries, EV-A71 infection is very common. Children of very young age are particularly vulnerable. Large-scale epidemics that occur every 3 to 4 years are associated with accumulation of an immunologically naive younger population. Capsid proteins especially VP1 with the presence of major B- and T-cell epitopes are the most antigenic proteins. The nonstructural proteins mainly contribute to T-cell epitopes that induce cross-reactive immune responses against other enteroviruses. Dominant epitopes and their neutralization magnitudes differ in mice, rabbits, and humans. Neutralizing antibody is sufficient for immune protection, but poorer cellular immunity may lead to severe neurological complications and deaths. Some chemokines/cytokines are consistently found in severely ill patients, for example, IL-6, IL-10, IL-17A, MCP-1, IL-8, MIG, IP-10, IFN-γ, and G-CSF. An increase in white cell counts is a risk factor for severe HFMD. Recent clinical trials on EV-A71 inactivated vaccine showed >90% efficacy and a robust neutralization response that was protective, indicating neutralizing antibody correlates for protection. No protection against other enteroviruses was observed. A comprehensive understanding of the immune responses to EV-A71 infection will benefit the development of diagnostic tools, potential therapeutics, and subunit vaccine candidates. Future development of a multivalent enterovirus vaccine will require knowledge of correlates of protection, understanding of cross-protection and memory T-cell responses among enteroviruses.
Thrombospondin-related adhesive protein (TRAP) of malaria parasites is essential for sporozoite motility and invasions into mosquito's salivary gland and vertebrate's hepatocyte; thereby, it is a promising target for pre-erythrocytic vaccine. TRAP of Plasmodium vivax (PvTRAP) exhibits sequence heterogeneity among isolates, an issue relevant to vaccine development. To gain insights into variation in the complete PvTRAP sequences of parasites in Thailand, 114 vivax malaria patients were recruited in 2006-2007 from 4 major endemic provinces bordering Myanmar (Tak in the northwest, n = 30 and Prachuap Khirikhan in the southwest, n = 25), Cambodia (Chanthaburi in the east, n = 29) and Malaysia (Yala and Narathiwat in the south, n = 30). In total, 26 amino acid substitutions were detected and 9 of which were novel, resulting in 44 distinct haplotypes. Haplotype and nucleotide diversities were lowest in southern P. vivax population while higher levels of diversities were observed in other populations. Evidences of positive selection on PvTRAP were demonstrated in domains II and IV and purifying selection in domains I, II and VI. Genetic differentiation was significant between each population except that between populations bordering Myanmar where transmigration was common. Regression analysis of pairwise linearized Fst and geographic distance suggests that P. vivax populations in Thailand have been isolated by distance. Sequence diversity of PvTRAP seems to be temporally stable over one decade in Tak province based on comparison of isolates collected in 1996 (n = 36) and 2006-2007. Besides natural selection, evidences of intragenic recombination have been supported in this study that could maintain and further generate diversity in this locus. It remains to be investigated whether amino acid substitutions in PvTRAP could influence host immune responses although several predicted variant T cell epitopes drastically altered the epitope scores. Knowledge on geographic diversity in PvTRAP constitutes an important basis for vaccine design provided that vaccination largely confers variant-specific immunity.
Plasmodium knowlesi is a simian malaria parasite that has been identified to cause malaria in humans. To date, several thousand cases of human knowlesi malaria have been reported around Southeast Asia. Thus far, there is no detailed study on genetic diversity and natural selection of P. knowlesi circumsporozoite protein (CSP), a prominent surface antigen on the sporozoite of the parasite. In the present study, the genetic diversity and natural selection acting on the nonrepeat regions of the gene encoding P. knowlesi CSP were investigated, focusing on the T-cell epitope regions at the C-terminal of the protein.
Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.
The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally devastated public health, the economies of many countries and quality of life universally. The recent emergence of immune-escaped variants and scenario of vaccinated individuals being infected has raised the global concerns about the effectiveness of the current available vaccines in transmission control and disease prevention. Given the high rate mutation of SARS-CoV-2, an efficacious vaccine targeting against multiple variants that contains virus-specific epitopes is desperately needed. An immunoinformatics approach is gaining traction in vaccine design and development due to the significant reduction in time and cost of immunogenicity studies and increasing reliability of the generated results. It can underpin the development of novel therapeutic methods and accelerate the design and production of peptide vaccines for infectious diseases. Structural proteins, particularly spike protein (S), along with other proteins have been studied intensively as promising coronavirus vaccine targets. Numbers of promising online immunological databases, tools and web servers have widely been employed for the design and development of next generation COVID-19 vaccines. This review highlights the role of immunoinformatics in identifying immunogenic peptides as potential vaccine targets, involving databases, and prediction and characterization of epitopes which can be harnessed for designing future coronavirus vaccines.
Considerable effort is directed at the development of a malaria vaccine that elicits antigen-specific T-cell responses against pre-erythrocytic antigens of Plasmodium falciparum. Genetic restriction of host T-cell responses and polymorphism of target epitopes on parasite antigens pose obstacles to the development of such a vaccine. Liver stage-specific antigen-1 (LSA-1) is a prime candidate vaccine antigen and five T-cell epitopes that are degenerately restricted by HLA molecules common in most populations have been identified on LSA-1. To define the extent of polymorphism within these T-cell epitopes, the N-terminal non-repetitive region of the LSA-1 gene from Malaysian P. falciparum field isolates was sequenced and compared with data of isolates from Brazil, Kenya and Papua New Guinea. Three of the T-cell epitopes were completely conserved while the remaining two were highly conserved in the isolates examined. Our findings underscore the potential of including these HLA-degenerate T-cell epitopes of LSA-1 in a subunit vaccine.
Autoreactivity to myeloperoxidase (MPO) causes anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), with rapidly progressive glomerulonephritis. Here, we show that a Staphylococcus aureus peptide, homologous to an immunodominant MPO T-cell epitope (MPO409-428), can induce anti-MPO autoimmunity. The peptide (6PGD391-410) is part of a plasmid-encoded 6-phosphogluconate dehydrogenase found in some S. aureus strains. It induces anti-MPO T-cell autoimmunity and MPO-ANCA in mice, whereas related sequences do not. Mice immunized with 6PGD391-410, or with S. aureus containing a plasmid expressing 6PGD391-410, develop glomerulonephritis when MPO is deposited in glomeruli. The peptide induces anti-MPO autoreactivity in the context of three MHC class II allomorphs. Furthermore, we show that 6PGD391-410 is immunogenic in humans, as healthy human and AAV patient sera contain anti-6PGD and anti-6PGD391-410 antibodies. Therefore, our results support the idea that bacterial plasmids might have a function in autoimmune disease.
The JCV (John Cunningham Virus) is known to cause progressive multifocal leukoencephalopathy, a condition that results in the formation of tumors. Symptoms of this condition such as sensory defects, cognitive dysfunction, muscle weakness, homonosapobia, difficulties with coordination, and aphasia. To date, there is no specific and effective treatment to completely cure or prevent John Cunningham polyomavirus infections. Since the best way to control the disease is vaccination. In this study, the immunoinformatic tools were used to predict the high immunogenic and non-allergenic B cells, helper T cells (HTL), and cytotoxic T cells (CTL) epitopes from capsid, major capsid, and T antigen proteins of JC virus to design the highly efficient subunit vaccines. The specific immunogenic linkers were used to link together the predicted epitopes and subjected to 3D modeling by using the Robetta server. MD simulation was used to confirm that the newly constructed vaccines are stable and properly fold. Additionally, the molecular docking approach revealed that the vaccines have a strong binding affinity with human TLR-7. The codon adaptation index (CAI) and GC content values verified that the constructed vaccines would be highly expressed in E. coli pET28a (+) plasmid. The immune simulation analysis indicated that the human immune system would have a strong response to the vaccines, with a high titer of IgM and IgG antibodies being produced. In conclusion, this study will provide a pre-clinical concept to construct an effective, highly antigenic, non-allergenic, and thermostable vaccine to combat the infection of the John Cunningham virus.
Enterovirus 71 (EV-71) is the main causative agent of hand, foot, and mouth disease (HFMD) which is generally regarded as a mild childhood disease. In recent years, EV71 has emerged as a significant pathogen capable of causing high mortalities and severe neurological complications in large outbreaks in Asia. A formalin-inactivated EV71 whole virus vaccine has completed phase III trial in China but is currently unavailable clinically. The high cost of manufacturing and supply problems may limit practical implementations in developing countries. Synthetic peptides representing the native primary structure of the viral immunogen which is able to elicit neutralizing antibodies can be made readily and is cost effective. However, it is necessary to conjugate short synthetic peptides to carrier proteins to enhance their immunogenicity. This review describes the production of cross-neutralizing anti-peptide antibodies in response to immunization with synthetic peptides selected from in silico analysis, generation of B-cell epitopes of EV71 conjugated to a promiscuous T-cell epitope from Poliovirus, and evaluation of the neutralizing activities of the anti-peptide antibodies. Besides neutralizing EV71 in vitro, the neutralizing antibodies were cross-reactive against several Enteroviruses including CVA16, CVB4, CVB6, and ECHO13.
Discovery of tumor antigenic epitopes is important for cancer vaccine development. Such epitopes can be designed and modified to become more antigenic and immunogenic in order to overcome immunosuppression towards the native tumor antigen. In silico-guided modification of epitope sequences allows predictive discrimination of those that may be potentially immunogenic. Therefore, only candidates predicted with high antigenicity will be selected, constructed, and tested in the lab. Here, we described the employment of in silico tools using a multiparametric approach to assess both potential T-cell epitopes (MHC class I/II binding) and B-cell epitopes (hydrophilicity, surface accessibility, antigenicity, and linear epitope). A scoring and ranking system based on these parameters was developed to shortlist potential mimotope candidates for further development as peptide cancer vaccines.
Dengue virus (DENV) comprises four serotypes (DENV1-4) which cause 390 million global infections with 500,000 hospitalizations and 25,000 fatalities annually. Currently, the only FDA approved DENV vaccine is the chimeric live-attenuated vaccine, Dengvaxia®, which is based on the yellow fever virus (YFV) genome that carries the prM and E genes of the respective DENV 1, 2, 3, and 4 serotypes. However, it has lower efficacies against serotypes DENV1 (51%) and DENV2 (34%) when compared with DENV3 (75%) and DENV4 (77%). The absence of T cell epitopes from non-structural (NS) and capsid (C) proteins of the yellow fever vaccine strain might have prevented Dengvaxia® to elicit robust cellular immune responses, as CD8+ T cell epitopes are mainly localized in the NS3 and NS5 regions. Multi-epitope-based peptide vaccines carrying CD4+, CD8+ T cell and B cell epitopes represent a novel approach to generate specific immune responses. Therefore, assessing and selecting epitopes that can induce robust B and T cell responses is a prerequisite for constructing an efficient multi-epitope peptide vaccine. Potent B and T cell epitopes can be identified by utilizing immunoinformatic analysis, but the immunogenicity of the epitopes have to be experimentally validated. In this review, we presented T cell epitopes that have been predicted by bioinformatic approaches as well as recent experimental validations of CD4+ and CD8+ T cell epitopes by ex-vivo stimulation of PBMCs with specific peptides. Immunoproteomic analysis could be utilized to uncover HLA-specific epitopes presented by DENV-infected cells. Based on various approaches, immunodominant epitopes capable of inducing strong immune responses could be selected and incorporated to form a universally applicable multi-epitope-based peptide dengue vaccine.
The genome feature of SARS-CoV-2 leads the virus to mutate and creates new variants of concern. Tackling viral mutations is also an important challenge for the development of a new vaccine. Accordingly, in the present study, we undertook to identify B- and T-cell epitopes with immunogenic potential for eliciting responses to SARS-CoV-2, using computational approaches and its tailoring to coronavirus variants. A total of 47 novel epitopes were identified as immunogenic triggering immune responses and no toxic after investigation with in silico tools. Furthermore, we found these peptide vaccine candidates showed a significant binding affinity for MHC I and MHC II alleles in molecular docking investigations. We consider them to be promising targets for developing peptide-based vaccines against SARS-CoV-2. Subsequently, we designed two efficient multi-epitopes vaccines against the SARS-CoV-2, the first one based on potent MHC class I and class II T-cell epitopes of S (FPNITNLCPF-NYNYLYRLFR-MFVFLVLLPLVSSQC), M (MWLSYFIASF-GLMWLSYFIASFRLF), E (LTALRLCAY-LLFLAFVVFLLVTLA), and N (SPRWYFYYL-AQFAPSASAFFGMSR). The second candidate is the result of the tailoring of the first designed vaccine according to three classes of SARS-CoV-2 variants. Molecular docking showed that the protein-protein binding interactions between the vaccines construct and TLR2-TLR4 immune receptors are stable complexes. These findings confirmed that the final multi-epitope vaccine could be easily adapted to new viral variants. Our study offers a shortlist of promising epitopes that can accelerate the development of an effective and safe vaccine against the virus and its adaptation to new variants.Communicated by Ramaswamy H. Sarma.
Jamestown Canyon virus (JCV) is a deadly viral infection transmitted by various mosquito species. This mosquito-borne virus belongs to Bunyaviridae family, posing a high public health threat in the in tropical regions of the United States causing encephalitis in humans. Common symptoms of JCV include fever, headache, stiff neck, photophobia, nausea, vomiting, and seizures. Despite the availability of resources, there is currently no vaccine or drug available to combat JCV. The purpose of this study was to develop an epitope-based vaccine using immunoinformatics approaches. The vaccine aimed to be secure, efficient, bio-compatible, and capable of stimulating both innate and adaptive immune responses. In this study, the protein sequence of JCV was obtained from the NCBI database. Various bioinformatics methods, including toxicity evaluation, antigenicity testing, conservancy analysis, and allergenicity assessment were utilized to identify the most promising epitopes. Suitable linkers and adjuvant sequences were used in the design of vaccine construct. 50s ribosomal protein sequence was used as an adjuvant at the N-terminus of the construct. A total of 5 CTL, 5 HTL, and 5 linear B cell epitopes were selected based on non-allergenicity, immunological potential, and antigenicity scores to design a highly immunogenic multi-peptide vaccine construct. Strong interactions between the proposed vaccine and human immune receptors, i.e., TLR-2 and TLR-4, were revealed in a docking study using ClusPro software, suggesting their possible relevance in the immunological response to the vaccine. Immunological and physicochemical properties assessment ensured that the proposed vaccine demonstrated high immunogenicity, solubility and thermostability. Molecular dynamics simulations confirmed the strong binding affinities, as well as dynamic and structural stability of the proposed vaccine. Immune simulation suggest that the vaccine has the potential to effectively stimulate cellular and humoral immune responses to combat JCV infection. Experimental and clinical assays are required to validate the results of this study.
Shigellosis is one of the most common diseases found in the developing countries, especially those countries that are prone flood. The causative agent for this disease is the Shigella species. This organism is one of the third most common enteropathogens responsible for childhood diarrhea. Since Shigella can survive gastric acidity and is an intracellular pathogen, it becomes difficult to treat. Also, uncontrolled use of antibiotics has led to development of resistant strains which poses a threat to public health. Therefore, there is a need for long term control of Shigella infection which can be achieved by designing a proper and effective vaccine. In this study, emphasis was made on designing a candidate that could elicit both B-cell and T-cell immune response. Hence B- and T-cell epitopes of outer membrane channel protein (OM) and putative lipoprotein (PL) from S. flexneri 2a were computationally predicted using immunoinformatics approach and a chimeric construct (chimeric-OP) containing the immunogenic epitopes selected from OM and PL was designed, cloned and expressed in E. coli system. The immunogenicity of the recombinant chimeric-OP was assessed using Shigella antigen infected rabbit antibody. The result showed that the chimeric-OP was a synthetic peptide candidate suitable for the development of vaccine and immunodiagnostics against Shigella infection.