Immunoinformatic evaluation of multiple epitope ensembles as vaccine candidates: E coli 536

Epitope prediction is becoming a key tool for vaccine discovery. Prospective analysis of bacterial and viral genomes can identify antigenic epitopes encoded within individual genes that may act as effective vaccines against specific pathogens. Since B-cell epitope prediction remains unreliable, we concentrate on T-cell epitopes, peptides which bind with high affinity to Major Histacompatibility Complexes (MHC). In this report, we evaluate the veracity of identified T-cell epitope ensembles, as generated by a cascade of predictive algorithms (SignalP, Vaxijen, MHCPred, IDEB, EpiJen), as a candidate vaccine against the model pathogen uropathogenic gram negative bacteria Escherichia coli (E-coli) strain 536 (O6:K15:H31). An immunoinformatic approach was used to identify 23 epitopes within the E-coli proteome. These epitopes constitute the most promiscuous antigenic sequences that bind across more than one HLA allele with high affinity (IC50 < 50nM). The reliability of software programmes used, polymorphic nature of genes encoding MHC and what this means for population coverage of this potential vaccine are discussed.     

Identification of a MHC class-II restricted epitope in carcinoembryonic antigen

Purpose The carcinoembryonic antigen (CEA) is extensively expressed on the vast majority of colorectal, gastric, and pancreatic carcinomas, and, therefore, is a good target for tumor immunotherapy. CD4+ T-helper (Th) cells play a critical role in initiation, regulation, and maintenance of immune responses. In this study, we sought to identify Th epitopes derived from CEA which can induce CEA-specific Th responses. The combined application with cytotoxic T lymphocyte (CTL) epitopes would be more potent than tumor vaccines that primarily activate CTL alone.Methods We utilized a combined approach of using a computer-based algorithm analysis TEPITOPE and in vitro biological analysis to identify Th epitopes in CEA.Results Initial screening of healthy donors showed that all five predicted peptides derived from CEA could induce peptide-specific T-cell proliferation in vitro. We characterized these CEA epitopes by establishing and analyzing peptide-specific T-cell clones. It was shown that CD4+ T-cells specific for the CEA₁₁₆ epitope can recognize and respond to naturally processed CEA protein and CEA₁₁₆ epitope can be promiscuously presented by commonly found major histocompatibility complex (MHC) alleles. Furthermore, it was demonstrated that immunization of human leukocyte antigen (HLA)-DR4 transgenic mice with CEA₁₁₆ peptide elicited antigen-specific Th responses which can recognize the antigenic peptides derived from CEA protein and CEA-positive tumors.Conclusion The MHC class II-restricted epitope CEA₁₁₆ could be used in the design of peptide-based tumor vaccine against several common cancers expressing CEA.     

Development of lipid-core-peptide (LCP) based vaccines for the prevention of group A streptococcal (GAS) infection

Traditional vaccines consisting of whole attenuated micro-organisms, or microbial components administered with adjuvant, have been demonstrated as one of the most cost-effective and successful public health interventions. Their use in large scale immunisation programs has lead to the eradication of smallpox, reduced morbidity and mortality from many once common diseases, and reduced strain on health services. However, problems associated with these vaccines including risk of infection, adverse effects, and the requirement for refrigerated transport and storage have led to the investigation of alternative vaccine technologies. Peptide vaccines, consisting of either whole proteins or individual peptide epitopes, have attracted much interest, as they may be synthesised to high purity and induce highly specific immune responses. However, problems including difficulties stimulating long lasting immunity, and population MHC diversity necessitating multiepitopic vaccines and/or HLA tissue typing of patients complicate their development. Furthermore, toxic adjuvants are necessary to render them immunogenic, and as such non-toxic human-compatible adjuvants need to be developed. Lipidation has been demonstrated as a human compatible adjuvant for peptide vaccines. The lipid-core-peptide (LCP) system, incorporating lipid adjuvant, carrier, and peptide epitopes, exhibits promise as a lipid-based peptide vaccine adjuvant. The studies reviewed herein investigate the use of the LCP system for developing vaccines to protect against group A streptococcal (GAS) infection. The studies demonstrate that LCP-based GAS vaccines are capable of inducing high-titres of antigen specific IgG antibodies. Furthermore, mice immunised with an LCP-based GAS vaccine were protected against challenge with 8830 strain GAS.     

Immunoinformatic Identification of Potential Epitopes Against Shigellosis

Diarrhoeal diseases due to Shigellosis account for deaths of ~1.5 million children every year in developing countries. Outer membrane proteins (OMPs) of Gram negative bacteria have been shown to be excellent subunit vaccine candidates against various pathogens. However, effective immune response can be generated using specific immunogenic determinants or peptides instead of whole protein or pathogen. In the present study, we chose six OMPs of Shigella flexneri 2a to predict peptides with good antigenic potential. Various tools were used in a systematic flow to predict B- and T-cell epitopes. Stringent selection criteria were used for epitope screening to ensure generation of both arms of immunity. These epitopes are predicted to be effective against a significantly large population of the diarrhoea afflicted countries in Southeast Asia. Most of the predicted epitopes are located towards the outer exposed region of proteins. The epitopes were docked with respective MHC Class I and II molecules to study peptide–MHC interactions. In conclusion, we have predicted an epitope ensemble against Shigellosis which can be experimentally validated for its immunogenic efficacy. We also propose a systematic workflow for immune-optimization to design effective peptide vaccines.     

Induction of protective and therapeutic antitumour immunity using a novel tumour-associated antigen-specific DNA vaccine

DNA vaccination has become an attractive immunization strategy against cancer. However, a major problem of DNA vaccination is its limited potency to be taken up by the antigen-presenting cells. In contrast, loss of immunogenic epitopes of tumour cells has urged the development of vaccines against multiple epitopes. In this study, we developed a novel strategy for the APC to efficiently cross-present a fusion tumour antigen, which contains both MHC class I-restricted and class II-restricted T-cell epitopes from Her-2/neu and p53 in a cognate manner. The N-terminus of the fusion Her-2/neu, p53 protein was linked to the sequence encoding for human secondary lymphoid-tissue chemokine for secretion and chemokinesis, and the C-terminus of the fusion protein was linked to a cell-binding domain of IgG (Fc portion, the cell-binding domain of IgG) for receptor-mediated internalization. Here, we show that the introduction of fused-gene DNA vaccine by gene gun reduced the size of established tumours and prolonged the lifespan of tumour-bearing mice. Results show that this DNA vaccination strategy can broadly enhance the antigen-specific cellular and humoral immune responses. This vaccine is capable of inducing adaptive immunity and may provide a novel, generic design for the development of therapeutic and preventive DNA vaccines.     

Selection of T-cell epitopes from foot-and-mouth disease virus reflects the binding affinity to different cattle MHC class II molecules

The major histocompatibility complex (MHC)-restricted selection of T-cell epitopes of foot-and-mouth disease virus (FMDV) by individual cattle MHC class II DR (BoLA-DR) molecules was studied in a direct MHC-peptide binding assay. By in vitro priming of T lymphocytes derived from animals homozygous for both MHC class I and II, five T-cell epitopes were analyzed in the context of three MHC class II haplotypes. We found that the presentation of these T-cell epitopes was mediated by DR molecules, since blocking this pathway of antigen presentation using monoclonal antibody TH14B completely abolished the proliferative responses against the peptides. To study the DR-restricted presentation of these T-cell epitopes, a direct MHC-peptide binding assay on isolated cattle DR molecules was developed. Purified cattle MHC class II DR molecules of the BoLA-DRB3*0201, BoLA-DRB3*1101, and BoLA-DRB3*1201 alleles were isolated from peripheral blood mononuclear cells. For each allele, one of the identified T-cell epitopes was biotinylated, and used as a marker peptide for the development of a competitive MHC-peptide binding assay. Subsequently, the T-cell epitopes of FMDV with functionally defined MHC class II specificity were analyzed in this binding assay. The affinity of the epitopes to bind to certain DR molecules was significantly correlated to the capacity to induce T-cell proliferation. This demonstrated at the molecular level that the selection of individual T-cell epitopes found at the functional level was indeed the result of MHC restriction.     

Prediction and molecular modeling of T-cell epitopes derived from placental alkaline phosphatase for use in cancer immunotherapy

In our ongoing efforts to combat cancer, peptide-based tumor vaccines are promising as one of the several alternatives used for cancer immunotherapy and immunoprevention. We have attempted to identify T-cell epitopes suitable for the development of a peptide-based cancer vaccine directed towards placental isozyme of alkaline phosphatase (PLAP), an oncofetal antigen. After identifying amino acid residues specific to PLAP and distinct from other close PLAP homologs, we have used sequence-based immunoinformatics tools (BIMAS and SYFPEITHI) and conducted molecular modeling studies using InsightII to investigate the binding affinity of the epitopes containing the unique residues with respective MHC class I molecules. Promiscuous epitopes binding to different alleles of different class I HLA loci were analyzed to get a population coverage that is widespread. Binding affinity deduced from the modeling studies corroborated the status of most of the epitopes scoring high in BIMAS and SYFPEITHI. We have thus identified specific epitopes from PLAP that have a potential for binding to their respective MHC class I alleles with high affinity. These peptides would be analysed in experiments to demonstrate their involvement in the induction of primary cytotoxic T-cell responses in vitro, using respective HLA-restricted T-cells in our way towards the development of an effective anti-cancer vaccine in a background of diverse MHC haplotypes.     

Sequential proteolytic processing of the capsular Caf1 antigen of Yersinia pestis for major histocompatibility complex class II-restricted presentation to T lymphocytes

We studied the mechanisms of antigen presentation of CD4 T cell epitopes of the capsular Caf1 antigen of Yersinia pestis using murine bone marrow macrophages as antigen presenting cells and T cell hybridomas specific for major histocompatibility complex (MHC) class II-restricted epitopes distributed throughout the Caf1 sequence. The data revealed diversity in the pathways used and the degrees of antigen processing required depending on the structural context of epitopes within the Caf1 molecule. Two epitopes in the carboxyl-terminal globular domain were presented by newly synthesized MHC class II after low pH-dependent lysosomal processing, whereas an epitope located in a flexible amino-terminal strand was presented by mature MHC class II independent of low pH and with no detectable requirement for proteolytic processing. A fourth epitope located between the two regions of Caf1 showed intermediate behavior. The data are consistent with progressive unfolding and cleavage of rCaf1 from the amino terminus as it traverses the endosomal pathway, the availability of epitopes determining which pool of MHC class II is preferentially loaded. The Caf1 capsular protein is a component of second generation plague vaccines and an understanding of the mechanisms and pathways of MHC class II-restricted presentation of multiple epitopes from this candidate vaccine antigen should inform the choice of delivery systems and adjuvants that target vaccines successfully to appropriate intracellular locations to induce protective immune responses against as wide a T cell repertoire as possible.     

Reconstruction of a pathway of antigen processing and class II MHC peptide capture

Endocytosed antigens are proteolytically processed and small amounts of peptides captured by class II MHC molecules. The details of antigen proteolysis, peptide capture and how destruction of T-cell epitopes is avoided are incompletely understood. Using the tetanus toxin antigen, we show that the introduction of 3-6 cleavage sites is sufficient to trigger a partially unfolded conformation able to bind to class II MHC molecules. The known locations of T-cell epitopes and protease cleavage sites predict that large domains of processed antigen (8-35 kDa) are captured under these conditions. Remarkably, when antigen is bound to the B-cell antigen receptor (BCR), processing can trigger a concerted 'hand-over' reaction whereby BCR-associated processed antigen is captured by neighbouring class II MHC molecules. Early capture of minimally processed antigen and confinement of the processing and class II MHC loading reaction to the membrane plane may improve the likelihood of T-cell epitope survival in the class II MHC pathway and may help explain the reciprocal relationships observed between B- and T-cell epitopes in many protein antigens and autoantigens.     

Prediction and Molecular Modeling of T-cell Epitopes Derived from Placental Alkaline Phosphatase for use in Cancer Immunotherapy

Abstract

In our ongoing efforts to combat cancer, peptide-based tumor vaccines are promising as one of the several alternatives used for cancer immunotherapy and immunoprevention. We have attempted to identify T-cell epitopes suitable for the development of a peptide-based cancer vaccine directed towards placental isozyme of alkaline phosphatase (PLAP), an oncofetal antigen. After identifying amino acid residues specific to PLAP and distinct from other close PLAP homologs, we have used sequence-based immunoinformatics tools (BIMAS and SYF- PEITHI) and conducted molecular modeling studies using InsightII to investigate the binding affinity of the epitopes containing the unique residues with respective MHC class I molecules. Promiscuous epitopes binding to different alleles of different class I HLA loci were analyzed to get a population coverage that is widespread. Binding affinity deduced from the modeling studies corroborated the status of most of the epitopes scoring high in BIMAS and SYFPEITHI. We have thus identified specific epitopes from PLAP that have a potential for binding to their respective MHC class I alleles with high affinity. These peptides would be analysed in experiments to demonstrate their involvement in the induction of primary cytotoxic T-cell responses in vitro, using respective HLA-restricted T-cells in our way towards the development of an effective anti-cancer vaccine in a background of diverse MHC haplotypes.     

Identification of B- and T-cell epitopes on HtrA protein of Orientia tsutsugamushi

Orientia tsutsugamushi, a cause of scrub typhus is emerging as an important pathogen in several parts of the tropics. The control of this infection relies on rapid diagnosis, specific treatment, and prevention through vector control. Development of a vaccine for human use would be very important as a public health measure. Antibody and T-cell response have been found to be important in the protection against scrub typhus. This study was undertaken to predict the peptide vaccine that elicits both B- and T-cell immunity. The outer-membrane protein, 47-kDa high-temperature requirement A was used as the target protein for the identification of protective antigen(s). Using BepiPred2 program, the potential B-cell epitope PNSSWGRYGLKMGLR with high conservation among O. tsutsugamushi and the maximum surface exposed residues was identified. Using IEDB, NetMHCpan, and NetCTL programs, T-cell epitopes MLNELTPEL and VTNGIISSK were identified. These peptides were found to have promiscuous class-I major histocompatibility complex (MHC) binding affinity to MHC supertypes and high proteasomal cleavage, transporter associated with antigen processing prediction, and antigenicity scores. In the I-TASSER generated model, the C-score was −0.69 and the estimated TM-score was 0.63 ± 0.14. The location of the epitope in the 3D model was external. Therefore, an antibody to this outer-membrane protein epitope could opsonize the bacterium for clearance by the reticuloendothelial system. The T-cell epitopes would generate T-helper function. The B-cell epitope(s) identified could be evaluated as antigen(s) in immunodiagnostic assays. This cocktail of three peptides would elicit both B- and T-cell immune response with a suitable adjuvant and serve as a vaccine candidate.     

Epitope-Based Vaccine Target Screening against Highly Pathogenic MERS-CoV: An In Silico Approach Applied to Emerging Infectious Diseases

Middle East respiratory syndrome coronavirus (MERS-CoV) with pandemic potential is a major worldwide threat to public health. However, vaccine development for this pathogen lags behind as immunity associated with protection is currently largely unknown. In this study, an immunoinformatics-driven genome-wide screening strategy of vaccine targets was performed to thoroughly screen the vital and effective dominant immunogens against MERS-CoV. Conservancy and population coverage analysis of the epitopes were done by the Immune Epitope Database. The results showed that the nucleocapsid (N) protein of MERS-CoV might be a better protective immunogen with high conservancy and potential eliciting both neutralizing antibodies and T-cell responses compared with spike (S) protein. Further, the B-cell, helper T-cell and cytotoxic T lymphocyte (CTL) epitopes were screened and mapped to the N protein. A total of 15 linear and 10 conformal B-cell epitopes that may induce protective neutralizing antibodies were obtained. Additionally, a total of 71 peptides with 9-mer core sequence were identified as helper T-cell epitopes, and 34 peptides were identified as CTL epitopes. Based on the maximum HLA binding alleles, top 10 helper T-cell epitopes and CTL epitopes that may elicit protective cellular immune responses against MERS-CoV were selected as MERS vaccine candidates. Population coverage analysis showed that the putative helper T-cell epitopes and CTL epitopes could cover the vast majority of the population in 15 geographic regions considered where vaccine would be employed. The B- and T-cell stimulation potentials of the screened epitopes is to be further validated for their efficient use as vaccines against MERS-CoV. Collectively, this study provides novel vaccine target candidates and may prompt further development of vaccines against MERS-CoV and other emerging infectious diseases.     

First peptide vaccine providing protection against viral infection in the target animal: studies of canine parvovirus in dogs.

A synthetic peptide vaccine which protects dogs against challenge with virulent canine parvovirus is described. The amino acid sequence used was discovered in previous studies on the immunogenic properties of previously mapped antigenic sites and represents the amino-terminal region of viral protein VP2. As with marker vaccines, it is possible to discriminate between vaccinated dogs that have not been exposed to the virus and dogs that have been infected with the virus. The protective mechanism can be explained by a humoral response against the peptide aided by T-cell epitopes contained in the carrier protein used for peptide coupling. This is the first example of a synthetic peptide vaccine that induces protection in target animals.     

A novel strategy of epitope design in Neisseria gonorrhoeae

In spite of genome sequences of both human and N. gonorrhoeae in hand, vaccine for gonorrhea is yet not available. Due to availability of several host and pathogen genomes and numerous tools for in silico prediction of effective B-cell and T-cell epitopes; recent trend of vaccine designing has been shifted to peptide or epitope based vaccines that are more specific, safe, and easy to produce. In order to design and develop such a peptide vaccine against the pathogen, we adopted a novel computational approache based on sequence, structure, QSAR, and simulation methods along with fold level analysis to predict potential antigenic B-cell epitope derived T-cell epitopes from four vaccine targets of N. gonorrhoeae previously identified by us [Barh and Kumar (2009) In Silico Biology 9, 1-7]. Four epitopes, one from each protein, have been designed in such a way that each epitope is highly likely to bind maximum number of HLA molecules (comprising of both the MHC-I and II) and interacts with most frequent HLA alleles (A*0201, A*0204, B*2705, DRB1*0101, and DRB1*0401) in human population. Therefore our selected epitopes are highly potential to induce both the B-cell and T-cell mediated immune responses. Of course, these selected epitopes require further experimental validation.     

HLA-A2-restricted T-cell epitopes specific for prostatic acid phosphatase

Prostatic acid phosphatase (PAP) has been investigated as the target of several antigen-specific anti-prostate tumor vaccines. The goal of antigen-specific active immunotherapies targeting PAP would ideally be to elicit PAP-specific CD8+ effector T cells. The identification of PAP-specific CD8+ T-cell epitopes should provide a means of monitoring the immunological efficacy of vaccines targeting PAP, and these epitopes might themselves be developed as vaccine antigens. In the current report, we hypothesized that PAP-specific epitopes might be identified by direct identification of pre-existing CD8+ T cells specific for HLA-A2-restricted peptides derived from PAP in the blood of HLA-A2-expressing individuals. 11 nonamer peptides derived from the amino acid sequence of PAP were used as stimulator antigens in functional ELISPOT assays with peripheral blood mononuclear cells from 20 HLA-A2+ patients with prostate cancer or ten healthy blood donors. Peptide-specific T cells were frequently identified in both groups for three of the peptides, p18–26, p112–120, and p135–143. CD8+ T-cell clones specific for three peptides, p18–26, p112–120, and p299–307, confirmed that these are HLA-A2-restricted T-cell epitopes. Moreover, HLA-A2 transgenic mice immunized with a DNA vaccine encoding PAP developed epitope-specific responses for one or more of these three peptide epitopes. We propose that this method to first identify epitopes for which there are pre-existing epitope-specific T cells could be used to prioritize MHC class I-specific epitopes for other antigens. In addition, we propose that the epitopes identified here could be used to monitor immune responses in HLA-A2+ patients receiving vaccines targeting PAP to identify potentially therapeutic immune responses.     

A structure-based approach for prediction of MHC-binding peptides

Identification of immunodominant peptides is the first step in the rational design of peptide vaccines aimed at T-cell immunity. The advances in sequencing techniques and the accumulation of many protein sequences without the purified protein challenge the development of computer algorithms to identify dominant T-cell epitopes based on sequence data alone. Here, we focus on antigenic peptides recognized by cytotoxic T cells. The selection of T-cell epitopes along a protein sequence is influenced by the specificity of each of the processing stages that precede antigen presentation. The most selective of these processing stages is the binding of the peptides to the major histocompatibility complex molecules, and therefore many of the predictive algorithms focus on this stage. Most of these algorithms are based on known binding peptides whose sequences have been used for the characterization of binding motifs or profiles. Here, we describe a structure-based algorithm that does not rely on previous binding data. It is based on observations from crystal structures that many of the bound peptides adopt similar conformations and placements within the MHC groove. The algorithm uses a structural template of the peptide in the MHC groove upon which peptide candidates are threaded and their fit to the MHC groove is evaluated by statistical pairwise potentials. It can rank all possible peptides along a protein sequence or within a suspected group of peptides, directing the experimental efforts towards the most promising peptides. This approach is especially useful when no previous peptide binding data are available.     

Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets

Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV) were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP), polymerase basic protein 1 (PB1) and matrix protein 1 (M1). C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks.     

Mimotopes of tumor-associated T-cell epitopes for cancer vaccines determined with combinatorial peptide libraries

Cytotoxic T-cells are the most important effector cells in immune responses against tumors. The identification of tumor-associated epitopes for these cells, therefore, has become a key aspect of the development of cancer vaccines. Here, we describe a new approach to the determination of tumor-associated T-cell epitopes which employs combinatorial peptide libraries with singly defined sequence positions in a randomized context. The analysis of the responses of a T-cell clone to these libraries yields the amino acid constituents of the epitope which can be combined to obtain mimotopes that are suitable as vaccine antigens for the induction of tumor-specific responses.     

The use of HPLC-MS in T-cell epitope identification

The hunt for T-cell epitopes is going on because hopes are set on such peptide sequences for diagnosis and vaccine development in the fight against infectious and tumor diseases. In addition to a variety of other techniques used in T-cell epitope identification, mass spectrometers coupled to microcapillary liquid chromatography have now become an important and sensitive tool in separation, detection, and sequence analysis of highly complex natural major histocompatibility complex (MHC) ligand mixtures. In this article, we review the basics of mass spectrometric techniques and their on-line coupling to microcapillary liquid chromatography (microcap-LC). Furthermore, we introduce current strategies for the identification of new T-cell epitopes using microcapillary liquid chromatography-mass spectrometry (microcap-LC-MS).     

Selective cytotoxic T-lymphocyte targeting of tumor immune escape variants

Defects in major histocompatibility complex (MHC) class I-restricted antigen presentation are frequently observed in human cancers and result in escape of tumors from cytotoxic T lymphocyte (CTL) immune surveillance in mice. Here, we show the existence of a unique category of CTLs that can prevent this escape. The CTLs target an alternative repertoire of peptide epitopes that emerge in MHC class I at the surface of cells with impaired function of transporter associated with antigen processing (TAP), tapasin or the proteasome. These peptides, although derived from self antigens such as the commonly expressed Lass5 protein (also known as Trh4), are not presented by normal cells. This explains why they act as immunogenic neoantigens. The newly discovered epitopes can be exploited for immune intervention against processing-deficient tumors through adoptive T-cell transfer or peptide vaccination.