In silico epitope identification of unique multidrug resistance proteins from Salmonella Typhi for vaccine development

Typhoid fever is a multisystemic illness caused by Salmonella enterica serovars Typhi and is resistant to most antibiotics and drugs. The resistance is conferred through multidrug resistance (MDR) proteins, which efflux most antibiotics and other drugs. We predicted potential candidate B-cell and T-cell epitopes using bio- and immune-informatics tools in the 11 MDR proteins - EmrA, EmrB, EmrD, MdtA, MdtB, MdtC, MdtG, MdtH, MdtK, MdtL and TolC. The antigenic potential of the MDR proteins was calculated using VaxiJen server. The B-cell and T-cell epitopes of the MDR proteins were predicted using BCPred and ProPredI and ProPred respectively. The binding affinities of the predicted T-cell epitopes were estimated using T-epitope designer and MHCPred tools. 10, 7, 5, 12, 14, 21, 26, 3, 3 and 3 B-cell epitopes were identified in EmrA, EmrB, EmrD, TolC, MdtA, MdtB, MdtC, MdtG, MdtH and MdtL respectively. We predicted 9 T-cell epitopes - YVSRRAVQP (EmrA), FGVANAISI (EmrB), MVNSQVKQA and YQGGMVNSQ (TolC), WDRTNSHKL (MdtA), FLRNIPTAI (MdtB), YVEQLGVTG (MdtG), VKWMYAIEA (MdtH) and LAHTNTVTL (MdtL) capable of eliciting both humoral and adaptive immune responses. These T-cell epitopes specifically bind to HLA alleles - DRB1*0101 and DRB1*0401. This is the first report of epitope prediction in the MDR proteins of S. Typhi. Taken together, these results indicate the MDR proteins – EmrA, MdtA and TolC are the most suitable vaccine candidates for S. Typhi. The findings of our study on the MDR proteins prove to be useful in the development of peptide-based vaccine for the prevention and/or treatment of typhoid fever.     

Modeling EBV infection and pathogenesis in new-generation humanized mice

The development of highly immunodeficient mouse strains has allowed the reconstitution of functional human immune system components in mice. New-generation humanized mice generated in this manner have been extensively used for modeling viral infections that are exclusively human tropic. Epstein–Barr virus (EBV)-infected humanized mice reproduce cardinal features of EBV-associated B-cell lymphoproliferative disease and EBV-associated hemophagocytic lymphohistiocytosis (HLH). Erosive arthritis morphologically resembling rheumatoid arthritis (RA) has also been recapitulated in these mice. Low-dose EBV infection of humanized mice results in asymptomatic, persistent infection. Innate immune responses involving natural killer cells, EBV-specific adaptive T-cell responses restricted by human major histocompatibility and EBV-specific antibody responses are also elicited in humanized mice. EBV-associated T-/natural killer cell lymphoproliferative disease, by contrast, can be reproduced in a distinct mouse xenograft model. In this review, recent findings on the recapitulation of human EBV infection and pathogenesis in these mouse models, as well as their application to preclinical studies of experimental anti-EBV therapies, are described.     

Designing an efficient multi-epitope peptide vaccine against Vibrio cholerae via combined immunoinformatics and protein interaction based approaches

Cholera continues to be a major global health concern. Among different Vibrio cholerae strains, only O1 and O139 cause acute diarrheal diseases that are related to epidemic and pandemic outbreaks. The currently available cholera vaccines are mainly lived and attenuated vaccines consisting of V. cholerae virulence factors such as toxin-coregulated pili (TCP), outer membrane proteins (Omps), and nontoxic cholera toxin B subunit (CTB). Nowadays, there is a great interest in designing an efficient epitope vaccine against cholera. Epitope vaccines consisting of immunodominant epitopes and adjuvant molecules enhance the possibility of inciting potent protective immunity. In this study, V. cholerae protective antigens (OmpW, OmpU, TcpA and TcpF) and the CTB, which is broadly used as an immunostimulatory adjuvant, were analyzed using different bioinformatics and immunoinformatics tools. The common regions between promiscuous epitopes, binding to various HLA-II supertype alleles, and B-cell epitopes were defined based upon the aforementioned protective antigens. The ultimately selected epitopes and CTB adjuvant were fused together using proper GPGPG linkers to enhance vaccine immunogenicity. A three-dimensional model of the thus constructed vaccine was generated using I-TASSER. The model was structurally validated using the ProSA-web error-detection software and the Ramachandran plot. The validation results indicated that the initial 3D model needed refinement. Subsequently, a high-quality model obtained after various refinement cycles was used for defining conformational B-cell epitopes. Several linear and conformational B-cell epitopes were determined within the epitope vaccine, suggesting likely antibody triggering features of our designed vaccine. Next, molecular docking was performed between the 3D vaccine model and the tertiary structure of the toll like receptor 2 (TLR2). To gain further insight into the interaction between vaccine and TLR2, molecular dynamics simulation was performed, corroborating stable vaccine-TLR2 binding. In sum, the results suggest that our designed epitope vaccine could incite robust long-term protective immunity against V. cholera.     

Two highly similar LAEDDTNAQKT and LTDKIGTEI epitopes in G glycoprotein may be useful for effective epitope based vaccine design against pathogenic Henipavirus

Nipah virus and Hendra virus, two members of the genus Henipavirus, are newly emerging zoonotic pathogens which cause acute respiratory illness and severe encephalitis in human. Lack of the effective antiviral therapy endorses the urgency for the development of vaccine against these deadly viruses. In this study, we employed various computational approaches to identify epitopes which has the potential for vaccine development. By analyzing the immune parameters of the conserved sequences of G glycoprotein using various databases and bioinformatics tools, we identified two potential epitopes which may be used as peptide vaccines. Using different B cell epitope prediction servers, four highly similar B cell epitopes were identified. Immunoinformatics analyses revealed that LAEDDTNAQKT is a highly flexible and accessible B-cell epitope to antibody. Highly similar putative CTL epitopes were analyzed for their binding with the HLA-C 12*03 molecule. Docking simulation assay revealed that LTDKIGTEI has significantly lower binding energy, which bolstered its potential as epitope-based vaccine design. Finally, cytotoxicity analysis has also justified their potential as promising epitope-based vaccine candidate. In sum, our computational analysis indicates that either LAEDDTNAQKT or LTDKIGTEI epitope holds a promise for the development of universal vaccine against all kinds of pathogenic Henipavirus. Further in vivo and in vitro studies are necessary to validate the obtained findings.     

In silico identification of outer membrane protein (Omp) and subunit vaccine design against pathogenic Vibrio cholerae

Virulence-related outer membrane proteins (Omps) are expressed in bacteria (Gram-negative) such as V. cholerae and are vital to bacterial invasion in to eukaryotic cell and survival within macrophages that could be best candidate for development of vaccine against V. cholerae. Applying in silico approaches, the 3-D model of the Omp was developed using Swiss model server and validated byProSA and Procheck web server. The continuous stretch of amino acid sequences 26 mer: RTRSNSGLLTWGDKQTITLEYGDPAL and 31 mer: FFAGGDNNLRGYGYKSISPQDASGALTGAKY having B-cell binding sites were selected from sequence alignment after B cell epitopes prediction by BCPred and AAP prediction modules of BCPreds. Further, the selected antigenic sequences (having B-cell epitopes) were analyzed for T-cell epitopes (MHC I and MHC II alleles binding sequence) by using ProPred 1 and ProPred respectively. The epitope (9 mer: YKSISPQDA) that binds to both the MHC classes (MHC I and MHC II) and covers maximum MHC alleles were identified. The identified epitopes can be useful in designing comprehensive peptide vaccine development against V. cholerae by inducing optimal immune response.     

Designing of precise vaccine construct against visceral leishmaniasis through predicted epitope ensemble: A contemporary approach

Visceral leishmaniasis (VL) caused by Leishmania donovani is a fatal parasitic disease affecting primarily the poor population in endemic countries. Increasing number of deaths as well as resistant to existing drugs necessitates the development of an effective vaccine for successful treatment of VL. The present study employed a combinatorial approach for designing monomer vaccine construct against L. donovani by applying forecasted B- and T- cell epitopes from 4 genome derived antigenic proteins having secretory signal peptides and glycophosphatidylinositol (GPI) anchors with ≤ 1 transmembrane helix. The forecasted population coverage of chosen T cell epitope ensemble (combined HLA class I and II) cover 99.14 % of world-wide human population. The predicted 3D structure of vaccine constructs (VC1/VC2) were modeled using homology modeling approach and docked to innate immune receptors TLR-2 and TLR-4 with respective docking energies −1231.4/−910.3 and −1119.4/−1476 kcal/mol. Overall, the aforementioned designed vaccine constructs were found appropriate for including in self-assembly protein nanoparticles (SAPN) for further study in developing cutting-edge precision vaccine against VL in short duration with cost-effective manner.     

An Epitope‐Specific Respiratory Syncytial Virus Vaccine Based on an Antibody Scaffold

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in children. We have generated an epitope‐specific RSV vaccine by grafting a neutralizing epitope (F‐epitope) in its native conformation into an immunoglobulin scaffold. The resulting antibody fusion exhibited strong binding affinity to Motavizumab, an RSV neutralizing antibody, and effectively induced potent neutralizing antibodies in mice. This work illustrates the potential of the immunoglobulin molecule as a scaffold to present conformationally constrained B‐cell epitopes.     

A novel in silico minigene vaccine based on CD4+ T-helper and B-cell epitopes of EG95 isolates for vaccination against cystic echinococcosis

EG95 oncospheral antigen plays a crucial role in Echinococcus granulosus pathogenicity. Considering the diversity of antigen among different EG95 isolates, it seems to be an ideal antigen for designing a universal multivalent minigene vaccine, so-called multi-epitope vaccine. This is the first in silico study to design a construct for the development of global EG95-based hydatid vaccine against E. granulosus in intermediate hosts. After antigen sequence selection, the three-dimensional structure of EG95 was modeled and multilaterally validated. The preliminary parameters for B-cell epitope prediction were implemented such as the possible transmembrane helix, signal peptide, post-translational modifications and allergenicity. The high ranked linear and conformational B-cell epitopes derived from several online web-servers (e.g., ElliPro, BepiPred v1.0, BcePred, ABCpred, SVMTrip, IEDB algorithms, SEPPA v2.0 and Discotope v2.0) were utilized for multiple sequence alignment and then for engineering the vaccine construct. T-helper based epitopes were predicted by molecular docking between the high frequent ovar class II allele (Ovar-DRB1*1202) and hexadecamer fragments of the EG95 protein. Having used the immune-informatics tools, we formulated the first EG95-based minigene vaccine based on T-helper epitope with high-binding affinity to the ovar MHC allele. This designed construct was analyzed for different physicochemical properties. It was also codon-optimized for high-level expression in Escherichia coli k12. Taken all, we propose the present in silico vaccine constructs as a promising platform for the generation of broadly protective vaccines for species and genus-specific immunization of the natural hosts of the parasite.     

Characterizing affinity epitopes between prion protein and β-amyloid using an epitope mapping immunoassay

Cellular prion protein, a membrane protein, is expressed in all mammals. Prion protein is also found in human blood as an anchorless protein, and this protein form is one of the many potential sources of misfolded prion protein replication during transmission. Many studies have suggested that β-amyloid_1–42 oligomer causes neurotoxicity associated with Alzheimer''s disease, which is mediated by the prion protein that acts as a receptor and regulates the hippocampal potentiation. The prevention of the binding of these proteins has been proposed as a possible preventative treatment for Alzheimer''s disease; therefore, a greater understanding of the binding hot-spots between the two molecules is necessary. In this study, the epitope mapping immunoassay was employed to characterize binding epitopes within the prion protein and complementary epitopes in β-amyloid. Residues 23–39 and 93–119 in the prion protein were involved in binding to β-amyloid_1–40 and _1–42, and monomers of this protein interacted with prion protein residues 93–113 and 123–166. Furthermore, β-amyloid antibodies against the C-terminus detected bound β-amyloid_1–42 at residues 23–40, 104–122 and 159–175. β-Amyloid epitopes necessary for the interaction with prion protein were not determined. In conclusion, charged clusters and hydrophobic regions of the prion protein were involved in binding to β-amyloid_1–40 and _1–42. The 3D structure appears to be necessary for β-amyloid to interact with prion protein. In the future, these binding sites may be utilized for 3D structure modeling, as well as for the pharmaceutical intervention of Alzheimer''s disease.     

Studies on peptides. CLXVII. Solid-phase syntheses and immunological properties of fragment peptides related to human malaria circumsporozoite protein

A glycine-linked tetramer of Asn-Ala-Asn-Pro, a tandem repeated sequence of malaria circumsporozoite (CS) protein, was synthesized by the Boc-based solid phase method, followed by deprotection with 1 M trimethylsilyl trifluoromethanesulfonate-thioanisole in trifluoroacetic acid. In addition, three tetramer-related peptides were similarly synthesized, i.e., a 34-residue peptide [linked with TH, a proposed T-cell epitope of CS, at the C-terminus of the tetramer], a 46-residue peptide and a 59-residue peptide [linked with HA or HA', two proposed T-cell epitopes of influenza hemagglutinin protein, at the N-terminus of the above 34-residue peptide]. Their immunological properties were examined by enzyme-linked immunosorbent assay, for which three different congenic strains of mouse were used to raise the specific antibodies. Despite conjugation of T-cell epitopes to the tetramer, the mice of low-responder strains to the tetramer failed to produce any antibody specific to the tetramer. However, with the aid of recombinant interleukin 2 as an adjuvant, the low-responder mice produced antibody with relatively high titers.     

An antibody reactive to the Gly63–Lys68 epitope of NT-proBNP exhibits O-glycosylation-independent binding

The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly₆₃–Lys₆₈ based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr₅₈ and Thr₇₁; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly₆₃–Lys₆₈ of NT-proBNP exhibits O-glycosylation-independent binding.     

A totally synthetic, self-assembling, adjuvant-free MUC1 glycopeptide vaccine for cancer therapy

In the development of vaccines for epithelial tumors, the key targets are MUC1 proteins, which have a variable number of tandem repeats (VNTR) bearing tumor-associated carbohydrate antigens (TACAs), such as Tn and STn. A major obstacle in vaccine development is the low immunogenicity of the short MUC1 peptide. To overcome this obstacle, we designed, synthesized, and evaluated several totally synthetic self-adjuvanting vaccine candidates with self-assembly domains. These vaccine candidates aggregated into fibrils and displayed multivalent B-cell epitopes under mild conditions. Glycosylation of Tn antigen on the Thr residue of PDTRP sequence in MUC1 VNTR led to effective immune response. These vaccines elicited a high level antibody response without any adjuvant and induced antibodies that recognized human breast tumor cells. These vaccines appeared to act through a T-cell independent pathway and were associated with the activation of cytotoxic T cells. These fully synthetic, molecularly defined vaccine candidates had several features that hold promise for anticancer therapy.     

Bodipy–C60 triple hydrogen bonding assemblies as heavy atom-free triplet photosensitizers: preparation and study of the singlet/triplet energy transfer

Supramolecular triplet photosensitizers based on hydrogen bonding-mediated molecular assemblies were prepared. Three thymine-containing visible light-harvesting Bodipy derivatives (B-1, B-2 and B-3, which show absorption at 505 nm, 630 nm and 593 nm, respectively) were used as H-bonding modules, and 1,6-diaminopyridine-appended C₆₀ was used as the complementary hydrogen bonding module (C-1), in which the C₆₀ part acts as a spin converter for triplet formation. Visible light-harvesting antennae with methylated thymine were prepared as references (B-1-Me, B-2-Me and B-3-Me), which are unable to form strong H-bonds with C-1. Triple H-bonds are formed between each Bodipy antenna (B-1, B-2 and B-3) and the C₆₀ module (C-1). The photophysical properties of the H-bonding assemblies and the reference non-hydrogen bond-forming mixtures were studied using steady state UV/vis absorption spectroscopy, fluorescence emission spectroscopy, electrochemical characterization, and nanosecond transient absorption spectroscopy. Singlet energy transfer from the Bodipy antenna to the C₆₀ module was confirmed by fluorescence quenching studies. The intersystem crossing of the latter produced the triplet excited state. The nanosecond transient absorption spectroscopy showed that the triplet state is either localized on the C₆₀ module (for assembly B-1·C-1), or on the styryl-Bodipy antenna (for assemblies B-2·C-1 and B-3·C-1). Intra-assembly forward–backward (ping-pong) singlet/triplet energy transfer was proposed. In contrast to the H-bonding assemblies, slow triplet energy transfer was observed for the non-hydrogen bonding mixtures. As a proof of concept, these supramolecular assemblies were used as triplet photosensitizers for triplet–triplet annihilation upconversion.     

Current status of multiple antigen-presenting peptide vaccine systems: Application of organic and inorganic nanoparticles

Many studies are currently investigating the development of safe and effective vaccines to prevent various infectious diseases. Multiple antigen-presenting peptide vaccine systems have been developed to avoid the adverse effects associated with conventional vaccines (i.e., live-attenuated, killed or inactivated pathogens), carrier proteins and cytotoxic adjuvants. Recently, two main approaches have been used to develop multiple antigen-presenting peptide vaccine systems: (1) the addition of functional components, e.g., T-cell epitopes, cell-penetrating peptides, and lipophilic moieties; and (2) synthetic approaches using size-defined nanomaterials, e.g., self-assembling peptides, non-peptidic dendrimers, and gold nanoparticles, as antigen-displaying platforms. This review summarizes the recent experimental studies directed to the development of multiple antigen-presenting peptide vaccine systems.     

A combination of epitope prediction and molecular docking allows for good identification of MHC class I restricted T-cell epitopes

In silico identification of T-cell epitopes is emerging as a new methodology for the study of epitope-based vaccines against viruses and cancer. In order to improve accuracy of prediction, we designed a novel approach, using epitope prediction methods in combination with molecular docking techniques, to identify MHC class I restricted T-cell epitopes. Analysis of the HIV-1 p24 protein and influenza virus matrix protein revealed that the present approach is effective, yielding prediction accuracy of over 80% with respect to experimental data. Subsequently, we applied such a method for prediction of T-cell epitopes in SARS coronavirus (SARS-CoV) S, N and M proteins. Based on available experimental data, the prediction accuracy is up to 90% for S protein. We suggest the use of epitope prediction methods in combination with 3D structural modelling of peptide-MHC-TCR complex to identify MHC class I restricted T-cell epitopes for use in epitope based vaccines like HIV and human cancers, which should provide a valuable step forward for the design of better vaccines and may provide in depth understanding about activation of T-cell epitopes by MHC binding peptides.     

Antibody synthesis induced by endogenous internal images

In this study, immunization with a vaccine consisting of multiple F(abt’)₂ fragments of affinity-purified antitetanus toxoid antibodies covalently bound to a carrier protein successfully induced antitetanus toxoid antibodies. Further studies showed that this vaccine preparation contained no biologically detectable tetanus antigen. The induced antitetanus antibody (Ab1t’) titer was higher than the titer of antibodies binding control antigens. The immunizing F(abt’)₂ preparation did not elicit a secondary antitetanus response from mice primed with tetanus toxoid and, hence, appeared free of tetanus epitopes. The specificity of Ab1t’ was established by absorption and inhibition with antigen. Immunization with antitetanus F(abt’)₂ (Ab1t’) fragments appears to have elicited naturally occurring autologous antitetanus toxoid antibody (Ab1t’) through an idiotypic pathway. As predicted by network theory, anti-idiotype (Ab2) and antitetanus (Ab1t’) cycled reciprocally. Clonotypic characterization of Ab1t’ using isoelectric focusing and affinity immunoblotting showed increases in Ab1t’ titer to be the result of increased synthesis by limited subsets of antitetanus toxoid B-cell clones and not increased synthesis by multiple clones, as is characteristic of antigen-driven Ab1 responses. Many Ab1 and Ab1t’ clonotypes had identical pIs, suggesting that they either share V region genes or are the product of the same B-cell clones. These findings indicate that immunization with polyclonal multivalent Ab1 preparations can trigger active synthesis of antibodies with the same specificity. The results provide further evidence for naturally occurring idiotypic cascades that could be exploited for studies of catalytic antibodies.     

Prediction of T-cell epitopes based on least squares support vector machines and amino acid properties

T-lymphocyte (T-cell) is a very important component in human immune system. It possesses a receptor (TCR) that is specific for the foreign epitopes which are in a form of short peptides bound to the major histocompatibility complex (MHC). When T-cell receives the message about the peptides bound to MHC, it makes the immune system active and results in the disposal of the immunogen. The antigenic determinants recognized and bound by the T-cell receptor is known as T-cell epitope. The accurate prediction of T-cell epitopes is crucial for vaccine development and clinical immunology. For the first time we developed new models using least squares support vector machine (LSSVM) and amino acid properties for T-cell epitopes prediction. A dataset including 203 short peptides (167 non-epitopes and 36 epitopes) was used as the input dataset and it was randomly divided into a training set and a test set. The models based on LSSVM and amino acid properties were evaluated using leave-one-out cross-validation method and the predictive ability of the test set, and obtained the results of 0.9875 and 0.9734 under the ROC curves, respectively. This result is more satisfactory than that were reported before. Especially, the accuracy of true positive gets a marked enhancement.     

Investigation into the Structural,Chemical and High Mechanical Reforms in B4C with Graphene Composite Material Substitution for Potential Shielding Frame Applications

In this work, boron carbide and graphene nanoparticle composite material (B₄C–G) was investigated using an experimental approach. The composite material prepared with the two-step stir casting method showed significant hardness and high melting point attributes. Scanning electron microscopy (SEM), along with energy dispersive X-ray spectroscopy (EDS) analysis, indicated 83.65%, 17.32%, and 97.00% of boron carbide + 0% graphene nanoparticles chemical compositions for the C-atom, Al-atom, and B₄C in the compound studied, respectively. The physical properties of all samples’ B₄C–G like density and melting point were 2.4 g/cm³ density and 2450 °C, respectively, while the grain size of B₄C–G was in the range of 0.8 ± 0.2 µm. XRD, FTIR, and Raman spectroscopic analysis was also performed to investigate the chemical compositions of the B₄C–G composite. The molding press composite machine was a fabrication procedure that resulted in the formation of outstanding materials by utilizing the sintering process, including heating and pressing the materials. For mechanical properties, high fracture toughness and tensile strength of B₄C–G composites were analyzed according to ASTM standard designs. The detailed analysis has shown that with 6% graphene content in B₄C, the composite material portrays a high strength of 134 MPa and outstanding hardness properties. Based on these findings, it is suggested that the composite materials studied exhibit novel features suitable for use in the application of shielding frames.     

Fully Synthetic Self‐Adjuvanting Thioether‐Conjugated Glycopeptide?Lipopeptide Antitumor Vaccines for the Induction of Complement‐Dependent Cytotoxicity against Tumor Cells

Glycopeptides of tumor‐associated mucin MUC1 are promising target structures for the development of antitumor vaccines. Because these endogenous structures were weakly immunogenic, they were coupled to immune‐response‐stimulating T‐cell epitopes and the Pam₃Cys lipopeptide to induce strong immune responses in mice. A new thioether‐ligation method for the synthesis of two‐ and three‐component vaccines that contain MUC1 glycopeptides as the B‐cell epitopes, a T‐cell epitope peptide, and the Pam₃CSK₄ lipopeptide is described. The resulting fully synthetic vaccines were used for the vaccination of mice, either in a liposome with Freund′s adjuvant or in aqueous PBS buffer. The three‐component vaccines that contained the Tetanus Toxoid P2 T‐cell epitope peptide induced strong immune responses, even when administered just in PBS. By activation of the complement‐dependent cytotoxicity (CDC) complex, the antisera induced the killing of tumor cells.     

A Fully Synthetic Four‐Component Antitumor Vaccine Consisting of a Mucin Glycopeptide Antigen Combined with Three Different T‐Helper‐Cell Epitopes

In a new concept of fully synthetic vaccines, the role of T‐helper cells is emphasized. Here, a synthetic antitumor vaccine consisting of a diglycosylated tumor‐associated MUC1 glycopeptide as the B‐cell epitope was covalently cross‐linked with three different T‐helper‐cell epitopes via squaric acid ligation of two linear (glyco)peptides. In mice this four‐component vaccine administered without external immune‐stimulating promoters elicit titers of MUC1‐specific antibodies that were about eight times higher than those induced by a vaccine containing only one T‐helper‐cell epitope. The promising results indicate that multiple activation of different T‐helper cells is useful for applications in which increased immunogenicity is required. In personalized medicine, in particular, this flexible construction of a vaccine can serve as a role model, for example, when T‐helper‐cell epitopes are needed that match human leukocyte antigens (HLA) in different patients.