Design Of Peptide-based Vaccines Research Articles

Epstein–Barr virus nuclear antigen 1‐specific CD4+ T cells directly kill Epstein–Barr virus‐carrying natural killer and T cells

Epstein-Barr virus (EBV) nuclear antigen (EBNA)1 is expressed in every EBV-infected cell, regardless of the state of EBV infection. Although EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies, it is less clear whether EBNA1-specific CD4(+) T cells can act as direct effectors. Herein, we investigated the ability of CD4(+) T-cell clones induced with overlapping peptides covering the C-terminal region of EBNA1, and identified minimal epitopes and their restricted major histocompatibility complex class II molecules. Of these, a novel epitope, EYHQEGGPD, was found to be presented by DRB1*0401, 0403 and 0406. Five CD4(+) T-cell clones recognized endogenously processed and presented antigens on EBV-transformed lymphoblastoid cell lines (LCL) and one example proved capable of killing EBV-carrying natural killer (NK) and T-cell lines derived from patients with chronic active EBV infection (CAEBV). Identification of minimal epitopes facilitates design of peptide-based vaccines and our data suggest that EBNA1-specific CD4(+) T cells may play roles as direct effectors for immunotherapy targeting EBV-carrying NK and T-cell malignancies.

Identification and Characterization of Dominant Helper T-Cell Epitopes in the Nucleocapsid Protein of Severe Acute Respiratory Syndrome Coronavirus

By using a series of overlapping synthetic peptides covering 98% of the amino acid sequence of the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus (SARS-CoV), four helper T-cell (Th) epitopes (NP11, residues 11 to 25; NP51, residues 51 to 65; NP61, residues 61 to 75; and NP111, residues 111 to 125) in C57BL mice (H-2(b)), four (NP21, residues 21 to 35; NP91, residues 91 to 105; NP331, residues 331 to 345; and NP351, residues 351 to 365) in C3H mice (H-2(k)), and two (NP81, residues 81 to 95; and NP351, residues 351 to 365) in BALB/c mice (H-2(d)) have been identified. All of these peptides were able to stimulate the proliferation of NP-specific T-cell lines or freshly isolated lymph node cells from mice immunized with recombinant NP. Immunization of mice with synthetic peptides containing appropriate Th epitopes elicited strong cellular immunity in vivo, as evidenced by delayed-type hypersensitivity. Priming with the helper peptides (e.g., NP111 and NP351) significantly accelerated the immune response induced by recombinant NP, as determined by the production of NP-specific antibodies. When fused with a conserved neutralizing epitope (SP1143-1157) from the spike protein of SARS-CoV, NP111 and NP351 assisted in the production of high-titer neutralizing antibodies in vivo. These data provide useful insights regarding immunity against SARS-CoV and have the potential to help guide the design of peptide-based vaccines.

POPI: predicting immunogenicity of MHC class I binding peptides by mining informative physicochemical properties

Both modeling of antigen-processing pathway including major histocompatibility complex (MHC) binding and immunogenicity prediction of those MHC-binding peptides are essential to develop a computer-aided system of peptide-based vaccine design that is one goal of immunoinformatics. Numerous studies have dealt with modeling the immunogenic pathway but not the intractable problem of immunogenicity prediction due to complex effects of many intrinsic and extrinsic factors. Moderate affinity of the MHC-peptide complex is essential to induce immune responses, but the relationship between the affinity and peptide immunogenicity is too weak to use for predicting immunogenicity. This study focuses on mining informative physicochemical properties from known experimental immunogenicity data to understand immune responses and predict immunogenicity of MHC-binding peptides accurately. This study proposes a computational method to mine a feature set of informative physicochemical properties from MHC class I binding peptides to design a support vector machine (SVM) based system (named POPI) for the prediction of peptide immunogenicity. High performance of POPI arises mainly from an inheritable bi-objective genetic algorithm, which aims to automatically determine the best number m out of 531 physicochemical properties, identify these m properties and tune SVM parameters simultaneously. The dataset consisting of 428 human MHC class I binding peptides belonging to four classes of immunogenicity was established from MHCPEP, a database of MHC-binding peptides (Brusic et al., 1998). POPI, utilizing the m = 23 selected properties, performs well with the accuracy of 64.72% using leave-one-out cross-validation, compared with two sequence alignment-based prediction methods ALIGN (54.91%) and PSI-BLAST (53.23%). POPI is the first computational system for prediction of peptide immunogenicity based on physicochemical properties. A web server for prediction of peptide immunogenicity (POPI) and the used dataset of MHC class I binding peptides (PEPMHCI) are available at http://iclab.life.nctu.edu.tw/POPI

Design of Peptide-Based Vaccines for Cancer

The immune system responds efficiently to bacteria, viruses and other agents however, the immune response to cancers is not as effective. In most cases other than specific genetic rearrangements leading to non-self proteins such as in leukemia and idiotypes in lymphoma, tumor associated proteins are self proteins and are not recognized by the immune system to prevent malignancy. In most cancers, patients develop antibodies and/or CTL-precursors to tumor associated antigens but are not effective in generating a therapeutic immune response. Adjuvants have been used with either whole tumors, subunits or peptides with the aim of increasing their immunity. Whole tumor antigens have certain advantages associated with it, such as ready availability as recombinant proteins, potential epitopes that can be presented by a number of MHC class I/II alleles and antibody development. The methods of identification of CD8 and CD4 epitopes either by use of epitope prediction algorithms or use of transgenic mice has made the use of defined synthetic peptides more attractive. The possibility to synthesize long peptides and introduce multiple epitopes (CD4 or CD8) from single or multiple antigens makes peptide a viable alternative to whole proteins. As an alternative to totally synthetic peptide constructs or polymers, polytopes have been generated by genetic engineering methods. In addition, to deliver immunogens to and to activate DC, receptor-mediated delivery of peptides using antibodies, cytokines and carbohydrates have been used. This review will encompass the various strategies, preclinical and clinical applications in designing peptide-based vaccines for cancer.

Narrowed TCR repertoire and viral escape as a consequence of heterologous immunity

Why some virus-specific CD8 TCR repertoires are diverse and others restricted or "oligoclonal" has been unknown. We show here that oligoclonality and extreme clonal dominance can be a consequence of T cell cross-reactivity. Lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV) encode NP(205-212) epitopes that induce different but highly cross-reactive diverse TCR repertoires. Homologous viral challenge of immune mice only slightly skewed the repertoire and enriched for predictable TCR motifs. However, heterologous viral challenge resulted in a narrow oligoclonal repertoire with dominant clones with unpredictable TCR sequences. This shift in clonal dominance varied with the private, i.e., unique, specificity of the host's TCR repertoire and was simulated using affinity-based computer models. The skewing differences in TCR repertoire following homologous versus heterologous challenge were observed within the same private immune system in mice adoptively reconstituted with memory CD8 T cell pools from the same donor. Conditions driving oligoclonality resulted in an LCMV epitope escape variant in vivo resembling the natural Lassa virus sequence. Thus, T cell oligoclonality, including extremes in clonal dominance, may be a consequence of heterologous immunity and lead to viral escape. This has implications for the design of peptide-based vaccines, which might unintentionally prime for skewed TCR responses to cross-reactive epitopes.

Predicting population coverage of T-cell epitope-based diagnostics and vaccines.

BackgroundT cells recognize a complex between a specific major histocompatibility complex (MHC) molecule and a particular pathogen-derived epitope. A given epitope will elicit a response only in individuals that express an MHC molecule capable of binding that particular epitope. MHC molecules are extremely polymorphic and over a thousand different human MHC (HLA) alleles are known. A disproportionate amount of MHC polymorphism occurs in positions constituting the peptide-binding region, and as a result, MHC molecules exhibit a widely varying binding specificity. In the design of peptide-based vaccines and diagnostics, the issue of population coverage in relation to MHC polymorphism is further complicated by the fact that different HLA types are expressed at dramatically different frequencies in different ethnicities. Thus, without careful consideration, a vaccine or diagnostic with ethnically biased population coverage could result.ResultsTo address this issue, an algorithm was developed to calculate, on the basis of HLA genotypic frequencies, the fraction of individuals expected to respond to a given epitope set, diagnostic or vaccine. The population coverage estimates are based on MHC binding and/or T cell restriction data, although the tool can be utilized in a more general fashion. The algorithm was implemented as a web-application available at .ConclusionWe have developed a web-based tool to predict population coverage of T-cell epitope-based diagnostics and vaccines based on MHC binding and/or T cell restriction data. Accordingly, epitope-based vaccines or diagnostics can be designed to maximize population coverage, while minimizing complexity (that is, the number of different epitopes included in the diagnostic or vaccine), and also minimizing the variability of coverage obtained or projected in different ethnic groups.

Insights into Peptide-Based Vaccine Design for Cancer Immunotherapy

The presentation of peptides derived from tumor associated proteins (TAAs) by the major histocompatibility complex (MHC) to T cell receptor (TcR) initiates a cascade of events that constitute the immune response. Eliciting an effective immune response, however, requires the coordinated regulation of both the cellular and humoral arms of the immune system. The design of effective peptide-based vaccines for cancer immunotherapeutic applications, therefore, requires intimate knowledge and understanding of peptide-MHC (pMHC) as well as TcR-pMHC interactions. Despite the wealth of information available to date from X-ray crystallographic and biological studies, the task of rationally designing peptide-based vaccines that can effectively prevent and/or treat cancer cell proliferation remains challenging. The complexity of interactions involved are not readily predictable and are further complicated by the involvement of surrounding molecules in vivo, which can lead to reduced biological activity and/or unwanted side effects. Furthermore, the delivery of peptide-based vaccines into the cell, for further processing and presentation to effector cell, represents an additional challenge which needs to be addressed. The incorporation of appropriate chemical entities into peptide-based vaccines can improve cellular uptake thereby enhancing biological activity. Finally, the susceptibility of peptide-based vaccines to enzymatic degradation warrants the need for the incorporation of non-natural amino acids, retro-inversion and/or cyclization to improve bioavailability essentially reducing the required dosage with minimum side effects.

Bcipep: A database of B-cell epitopes

BackgroundBcipep is a database of experimentally determined linear B-cell epitopes of varying immunogenicity collected from literature and other publicly available databases.ResultsThe current version of Bcipep database contains 3031 entries that include 763 immunodominant, 1797 immunogenic and 471 null-immunogenic epitopes. It covers a wide range of pathogenic organisms like viruses, bacteria, protozoa, and fungi. The database provides a set of tools for the analysis and extraction of data that includes keyword search, peptide mapping and BLAST search. It also provides hyperlinks to various databases such as GenBank, PDB, SWISS-PROT and MHCBN.ConclusionA comprehensive database of B-cell epitopes called Bcipep has been developed that covers information on epitopes from a wide range of pathogens. The Bcipep will be source of information for investigators involved in peptide-based vaccine design, disease diagnosis and research in allergy. It should also be a promising data source for the development and evaluation of methods for prediction of B-cell epitopes. The database is available at .

Rational Peptide-Based Vaccine Design for Cancer Immunotherapeutic Applications

Immune responses to cancer cells can be elicited in vivo by administering synthetic peptides derived from proteins uniquely or overexpressed on tumor cells (tumor associated antigens--TAAs). Peptides derived from TAAs are presented in the context of major histocompatibility complex (MHC) molecules to cytotoxic T cells (CTL), which can recognize and lyze tumor cells. In contrast to peptides derived from an exogenous source (viral or bacterial), tumor peptides bind weakly to MHC class I molecules. The low binding affinity of these peptides makes them poor candidates for vaccination due to the poor immunogenic response produced. In order to enhance antigen recognition and hence immunogenicity, peptide binding affinity for MHC can be initially improved by modifying the "anchor" residues. However, the task at hand is highly unpredictable and minor changes in peptide sequence can alter/abolish the T cell response. Furthermore, despite the wealth of information obtained over the last decade from high resolution X-ray structures of MHC class I in complex with peptides (pMHC) as well as pMHC in complex with T cell receptor (TcR), prediction remains difficult. Nonetheless, peptides represent convenient chemical entities that can be rapidly synthesized in clinical grade for therapeutic applications. Herein, the rationale behind modifying TAAs will be discussed including the synthesis/use of proteolytically tolerant peptides (and peptide mimetics) which incorporate non-natural amino acids, retro-inversion and cyclization to improve bioavailability.

Biochemical and structural impact of natural polymorphism in the HLA-A3 superfamily

Class I alleles of the HLA-A3 superfamily (-A*0301, -A*1101, -A*3101, -A*3301, and -Aw*6801) share largely overlapping peptide repertoires. Cross-reactive T cell responses between HLA-A3-like molecule/peptide complexes have been demonstrated in vitro and during natural diseases. In spite of this immune relatedness, HLA-A3-like molecules exhibit noticeable differences in their antigen-selecting and -presenting properties. Identifying molecular and structural features responsible for these differences is important for understanding how natural polymorphism leads to functional divergence within the HLA-A3 superfamily. Towards this goal, we used an approach that combines thermal stability data on recombinant, soluble HLA-A3-like molecules complexed with a nonamer and decamer HIV-1 peptide, together with a detailed structural analysis of these HLA-A3-like molecule/peptide complexes based on crystal and molecular model structures. Our studies revealed the importance of residues 9 and 67 for modulating peptide selection within the B pocket; of residue 97 for modulating peptide selection within the F pocket interdependently with the presence (or absence) of a middle, secondary anchor residue; and of residues 70, 73, 97, 152, and 156 for modulating peptide presentation in the central region of the groove that leads to altered antigenic surfaces. Overall, our detailed assessment of the biochemical and structural impact of natural polymorphism within the HLA-A3 superfamily has permitted to understand how HLA-A3-like molecules differ at the level of their primary and secondary anchor pockets causing fine differences in their peptide-selecting and -presenting properties. A better understanding of the molecular immunological properties of HLA-A3-like molecules is significantly important for the rationale design of broad peptide-based vaccines.

Virtual models of the HLA class I antigen processing pathway

Antigen recognition by cytotoxic CD8 T cells is dependent upon a number of critical steps in MHC class I antigen processing including proteosomal cleavage, TAP transport into the endoplasmic reticulum, and MHC class I binding. Based on extensive experimental data relating to each of these steps there is now the capacity to model individual antigen processing steps with a high degree of accuracy. This paper demonstrates the potential to bring together models of individual antigen processing steps, for example proteosome cleavage, TAP transport, and MHC binding, to build highly informative models of functional pathways. In particular, we demonstrate how an artificial neural network model of TAP transport was used to mine a HLA-binding database so as to identify HLA-binding peptides transported by TAP. This integrated model of antigen processing provided the unique insight that HLA class I alleles apparently constitute two separate classes: those that are TAP-efficient for peptide loading ( HLA-B27, -A3, and -A24) and those that are TAP-inefficient ( HLA-A2, -B7, and -B8). Hence, using this integrated model we were able to generate novel hypotheses regarding antigen processing, and these hypotheses are now capable of being tested experimentally. This model confirms the feasibility of constructing a virtual immune system, whereby each additional step in antigen processing is incorporated into a single modular model. Accurate models of antigen processing have implications for the study of basic immunology as well as for the design of peptide-based vaccines and other immunotherapies.

Peptide Based Vaccine Design for Cancer Immunotherapy

Peptide based vaccine design for cancer immunotherapy is currently being used in clinical trials for malignant melanoma with much success. Administration of synthetic peptides derived from proteins overexpressed in tumour cells (tumour-associated antigens) can elicit tumour-specific CD8+ T cell responses in vitro and in vivo. Currently, a number of tumour antigens from overexpressed cancer proteins such as mucin 1 (MUC1), survivin, carcinoembryonic antigen (CEA), telomerase reverse transcriptase (TERT) and HER- 2 / neu are examples of cancers whereby peptide based vaccinations are being tested for potential immunotherapy. In this review, we discuss some examples of the work being investigated with these cancer proteins, limitations to this therapy and suggestions of future directions to improve the efficacy of this treatment. Keywords: peptide, cancer immunotherapy, tumour associated antigens, cytotoxic t cells, muc1

Variation in vaccine response in normal populations.

Genetic polymorphisms of the human leukocyte antigen (HLA) system significantly influence the variation in immune responses to viral vaccines. Considerable data on the genetic determinants of immune responses to the measles vaccine support the importance of HLA genes in determining the variation in vaccine response. HLA class I and class II, TAP, and HLA-DM allele associations with measles-specific antibody levels following measles vaccination have revealed, in part, the immunologic basis for mechanisms of measles immunity variation. Associations between HLA genotype and immune responses have also been reported for other vaccines and infectious diseases, such as hepatitis B and C, human papillomaviruses, and influenza. Vaccine pharmacogenomics may provide important insights for the design and development of new peptide-based vaccines against measles and other pathogens.

Identification of immunodominant regions among promiscuous HLA-DR–restricted CD4+ T-cell epitopes on the tumor antigen MAGE-3

AbstractThe molecular characterization of the CD4+ T-cell epitope repertoire on human tumor antigens would contribute to both clinical investigation and cancer immunotherapy. In particular, the identification of promiscuous epitopes would be beneficial to a large number of patients with neoplastic diseases regardless of their HLA-DR type. MAGE-3 is a tumor-specific antigen widely expressed in solid and hematologic malignancies; therefore, is an excellent candidate antigen. We used a major histocompatability complex (MHC) class II epitope prediction algorithm, the TEPITOPE software, to predict 11 sequence segments of MAGE-3 that could form promiscuous CD4+ T-cell epitopes. In binding assays, the synthetic peptides corresponding to the 11 predicted sequences bound at least 3 different HLA-DR alleles. Nine of the 11 peptides induced proliferation of CD4+ T cells from both healthy subjects and melanoma patients. Four immunodominant regions (residues 111-125, 146-160, 191-205, and 281-295), containing naturally processed epitopes, were recognized by most of the donors, in association with 3 to 4 different HLA-DR alleles, thus covering up to 94% of the alleles expressed in whites. On the contrary, the other promiscuous regions (residues 161-175 and 171-185) contained epitopes not naturally processed in vitro. The immunodominant epitopes identified will be useful in the design of peptide-based cancer vaccines and in the study of the functional state of tumor-specific CD4+ T cells in patients bearing tumors expressing MAGE-3.

Crystal Structure of a Non-canonical Low-affinity Peptide Complexed with MHC Class I: A New Approach For Vaccine Design

Peptides bind with high affinity to MHC class I molecules by anchoring certain side-chains (anchors) into specificity pockets in the MHC peptide-binding groove. Peptides that do not contain these canonical anchor residues normally have low affinity, resulting in impaired pMHC stability and loss of immunogenicity. Here, we report the crystal structure at 1.6Å resolution of an immunogenic, low-affinity peptide from the tumor-associated antigen MUC1, bound to H-2Kb. Stable binding is still achieved despite small, non-canonical residues in the C and F anchor pockets. This MUC1/Kb structure reveals how low-affinity peptides can be utilized in the design of novel peptide-based tumor vaccines. The molecular interactions elucidated in this non-canonical low-affinity peptide MHC complex should help uncover additional immunogenic peptides from primary protein sequences and aid in the design of alternative approaches for T-cell vaccines.

The central role played by peptides in the immune response and the design of peptide-based vaccines against infectious diseases and cancer.

Vaccines are one of the most cost effective methods of improving public health thereby increasing the quality of life. Prophylactic and therapeutic treatment by vaccines can prevent infectious diseases and some cancers and could also be used in the treatment of autoimmune disorders. An appreciation of this potential has resulted in a burgeoning literature which not only describes the scientific efforts being made into designing new and improved vaccines but also drives the efforts being made by public health organizations world-wide in delivering vaccines to the community. At the forefront of technologies being applied to the design of vaccines is the use of synthetic peptides; the chemical technologies used to assemble peptides have made great strides over the last decade and assembly of hi-fidelity peptides which can be of high molecular weight, multimeric or even branched is now almost routine. Together with the advances in peptide technology our understanding of the molecular events that are necessary to induce immune responses has also made great strides. The central role that peptides play in immune recognition is now recognised and rules are emerging that are being applied to the construction of peptide-based vaccines that, in the right context, can induce humoral (antibody) and cellular (cytotoxic and helper T cell) immune responses. Synthetic peptides are exquisitely placed to answer questions about immune recognition and along the way to provide us with new and improved vaccines.

Peptide based vaccine design: Synthesis and immunological characterization of branched polypeptide conjugates comprising the 276–284 immunodominant epitope of HSV-1 glycoprotein D

Peptide based vaccine design: Synthesis and immunological characterization of branched polypeptide conjugates comprising the 276–284 immunodominant epitope of HSV-1 glycoprotein D

Peptide based vaccine design: Synthesis and immunological characterization of branched polypeptide conjugates comprising the 276–284 immunodominant epitope of HSV‐1 glycoprotein D

The importance of the length and conjugation site of a protective epitope peptide (276SALLEDPVG284) from glycoprotein D of herpes simplex virus in branched polypeptide conjugates has been investigated. A new set of peptides, with a single attachment site and truncated sequences, was prepared. The immunogenicity of conjugates and the specificity of antibody responses elicited were investigated in BALB/c, C57/B1/6 and CBA mice. It was found that the covalent coupling of the peptide comprising the 276-284 sequence of gD through its Asp residue at position 281 did not influence the immunogenic properties of the epitope, while involvement of the side chain of Glu at position 280 almost completely abolished immunogenicity. These results clearly indicated that the conjugation site of the epitope peptide influenced the intensity and specificity of antibody responses. Comparison of the immunological properties of conjugates containing truncated gD peptides revealed the presence of two epitopes within the 276-284 region. One of the proposed epitopes is situated at the N-terminal (276-281) region, while the other is located at the C-terminal end of the sequence (279-284). Binding data demonstrated that some of the peptides comprising these epitopes induced gD-specific responses in their conjugated form and also elicited an immune response that conferred protection against lethal HSV-1 infection. The correlation of peptide- and gD-specific antibody responses with the protective effect of the immune response is discussed.

Contribution of HLA Class I Allele Expression to CD8+ T‐Cell Responses against Epstein–Barr Virus

Most immune responses to viral infections involve CD8+ T cells recognizing viral peptides of typically 9-10 amino acids in the groove of major histocompatibility complex (MHC) class I. Importantly, CD8+ T-cell responses appear to focus on few viral epitopes, a phenomenon termed immunodominance. While the understanding of this phenomenon has been based largely on experimental mice models, it is imperative to evaluate its contribution in humans, as the design of peptide-based vaccines may be influenced by immunodomination. Here, we present evidence that immunodominance can be detected among Epstein-Barr virus (EBV) epitopes associated with two of the most frequent class I alleles in Western Europe, human leucocyte antigen (HLA)-A2 and HLA-B7. CD8+ T-cell responses to HLA-A2-associated EBV epitopes were significantly reduced in individuals coexpressing HLA-B7. The impairment of HLA-A2-associated responses correlated with a dominant response to an HLA-B7 epitope. The data demonstrate a hierarchy in the human cellular immune response to immunodominant EBV epitopes presented by separate HLA class I alleles. This may have implications for EBV vaccine development as well as for the interpretation of isolated analysis of immunodominant responses to EBV.

Rational design of peptide-based tumor vaccines.

Administration of synthetic peptides derived from proteins uniquely or overexpressed in tumor cells (tumor-associated antigens) can elicit tumor-specific immune responses in vivo. This is because cytotoxic T lymphocytes can recognize and lyse tumor cells that display peptides derived from tumor-associated antigens (TAAs) in the context of class I major histocompatibility complex (MHC) molecules. TAA peptides, in contrast to peptides of viral origin, generally bind weakly to the MHC molecule. In many cases, this explains the poor magnitude of T cell response directed at the tumor in vivo. Improving MHC binding as a strategy to upregulate antigen recognition can convert low affinity TAA peptides into useful tools in clinical trial settings. High-resolution structures of class I MHC molecules reported over the past two decades provided the framework for designing peptides that can induce optimal T cell response. This review will discuss the basic and clinical aspects of modifying native TAA peptides as tumor vaccines.