Applications In Vaccine Design Research Articles

CryoEM structure of an MHC-I/TAPBPR peptide bound intermediate reveals the mechanism of antigen proofreading.

Class I major histocompatibility complex (MHC-I) proteins play a pivotal role in adaptive immunity by displaying epitopic peptides to CD8+ T cells. The chaperones tapasin and TAPBPR promote the selection of immunogenic antigens from a large pool of intracellular peptides. Interactions of chaperoned MHC-I molecules with incoming peptides are transient in nature, and as a result, the precise antigen proofreading mechanism remains elusive. Here, we leverage a high-fidelity TAPBPR variant and conformationally stabilized MHC-I, to determine the solution structure of the human antigen editing complex bound to a peptide decoy by cryogenic electron microscopy (cryo-EM) at an average resolution of 3.0 Å. Antigen proofreading is mediated by transient interactions formed between the nascent peptide binding groove with the P2/P3 peptide anchors, where conserved MHC-I residues stabilize incoming peptides through backbone-focused contacts. Finally, using our high-fidelity chaperone, we demonstrate robust peptide exchange on the cell surface across multiple clinically relevant human MHC-I allomorphs. Our work has important ramifications for understanding the selection of immunogenic epitopes for T cell screening and vaccine design applications.

Synergistic Immunity and Protection in Mice by Co-Immunization with DNA Vaccines Encoding the Spike Protein and Other Structural Proteins of SARS-CoV-2.

The emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated recurring worldwide infection outbreaks. These highly mutated variants reduce the effectiveness of current coronavirus disease 2019 (COVID-19) vaccines, which are designed to target only the spike (S) protein of the original virus. Except for the S of SARS-CoV-2, the immunoprotective potential of other structural proteins (nucleocapsid, N; envelope, E; membrane, M) as vaccine target antigens is still unclear and worthy of investigation. In this study, synthetic DNA vaccines encoding four SARS-CoV-2 structural proteins (pS, pN, pE, and pM) were developed, and mice were immunized with three doses via intramuscular injection and electroporation. Notably, co-immunization with two DNA vaccines that expressed the S and N proteins induced higher neutralizing antibodies and was more effective in reducing the SARS-CoV-2 viral load than the S protein alone in mice. In addition, pS co-immunization with either pN or pE + pM induced a higher S protein-specific cellular immunity after three immunizations and caused milder histopathological changes than pS alone post-challenge. The role of the conserved structural proteins of SARS-CoV-2, including the N/E/M proteins, should be investigated further for their applications in vaccine design, such as mRNA vaccines.

Enhancement of immune responses using ovalbumin-conjugated Eucommia ulmoides leaf polysaccharides encapsulated in a cubic liquid-crystalline phase delivery system.

To improve the adjuvant activity of polysaccharides from Eucommia ulmoides leaves (PsEUL) in inducing an effective immune response against ovalbumin (OVA), PsEUL were conjugated to OVA using the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) method. The synthesized PsEUL-OVA was encapsulated using phytantriol and F127 to produce PsEUL-OVA cubosomes (Cubs), a novel delivery system. The physicochemical properties and immune modulation effects of this novel delivery system were explored. In vitro, PsEUL-OVA/Cubs carrying large amounts of OVA were rapidly phagocytized by macrophages and upregulated macrophage proliferation, thereby stimulating cytokine production (interleukin (IL)-6 and IL-4). In vivo, PsEUL-OVA/Cubs increased the titer of OVA-specific antibodies (immunoglobulin (Ig)G, IgG2b, IgG2a and IgG1) and cytokine levels (IL-2, IL-6, IL-4 and interferon-γ). In addition, the cubosomes promoted the differentiation of CD8+ and CD4+ T cells in the spleen and the maturation of dendritic cells (DCs). These results indicated that PsEUL-OVA/Cubs stimulated both cellular and humoral immune responses by enhancing the phagocytic activity of DCs and macrophages and increasing the antigen presentation efficiency. Collectively, the findings demonstrate that PsEUL-antigen/Cubs can be a useful delivery vehicle with immune response-promoting effects. Therefore, this study lays the foundation for the development of novel adjuvant-antigen delivery systems with potential applications in vaccine design. © 2022 Society of Chemical Industry.

Epitopepredict: a tool for integrated MHC binding prediction.

A key step in the cellular adaptive immune response is the presentation of antigens to T cells. Computational prediction of T cell epitopes has many applications in vaccine design and immuno-diagnostics. This is the basis of immunoinformatics, which allows in silico screening of peptides before experiments are performed. With the availability of whole genomes for many microbial species it is now feasible to computationally screen whole proteomes for candidate peptides. epitopepredict is a programmatic framework and command line tool designed to aid this process. It provides access to multiple binding prediction algorithms under a single interface and scales for whole genomes using multiple target MHC alleles. A web interface is provided to assist visualization and filtering of the results. The software is freely available under an open-source license from https://github.com/dmnfarrell/epitopepredict.

Designs of Antigen Structure and Composition for Improved Protein-Based Vaccine Efficacy.

Today, vaccinologists have come to understand that the hallmark of any protective immune response is the antigen. However, it is not the whole antigen that dictates the immune response, but rather the various parts comprising the whole that are capable of influencing immunogenicity. Protein-based antigens hold particular importance within this structural approach to understanding immunity because, though different molecules can serve as antigens, only proteins are capable of inducing both cellular and humoral immunity. This fact, coupled with the versatility and customizability of proteins when considering vaccine design applications, makes protein-based vaccines (PBVs) one of today's most promising technologies for artificially inducing immunity. In this review, we follow the development of PBV technologies through time and discuss the antigen-specific receptors that are most critical to any immune response: pattern recognition receptors, B cell receptors, and T cell receptors. Knowledge of these receptors and their ligands has become exceptionally valuable in the field of vaccinology, where today it is possible to make drastic modifications to PBV structure, from primary to quaternary, in order to promote recognition of target epitopes, potentiate vaccine immunogenicity, and prevent antigen-associated complications. Additionally, these modifications have made it possible to control immune responses by modulating stability and targeting PBV to key immune cells. Consequently, careful consideration should be given to protein structure when designing PBVs in the future in order to potentiate PBV efficacy.

Classification of self-assembling protein nanoparticle architectures for applications in vaccine design.

We introduce here a mathematical procedure for the structural classification of a specific class of self-assembling protein nanoparticles (SAPNs) that are used as a platform for repetitive antigen display systems. These SAPNs have distinctive geometries as a consequence of the fact that their peptide building blocks are formed from two linked coiled coils that are designed to assemble into trimeric and pentameric clusters. This allows a mathematical description of particle architectures in terms of bipartite (3,5)-regular graphs. Exploiting the relation with fullerene graphs, we provide a complete atlas of SAPN morphologies. The classification enables a detailed understanding of the spectrum of possible particle geometries that can arise in the self-assembly process. Moreover, it provides a toolkit for a systematic exploitation of SAPNs in bioengineering in the context of vaccine design, predicting the density of B-cell epitopes on the SAPN surface, which is critical for a strong humoral immune response.

HIV-1 Broadly Neutralizing Antibodies: Identification, Development and Vaccine Evaluation

Numerous human immunodeficiency virus-1 (HIV-1) broadly neutralizing antibodies (bNAbs) have been produced and details of their generation, evolution and structure provide a blueprint for effective vaccination. Recent advances in next generation sequencing (NGS) technologies have allowed high-resolution characterization of the antibody repertoire, and have enabled thorough characterization of many HIV-1 bNAbs. These bNAbs identified conserved epitopes that may be used to design new vaccines and provided new tools for prophylaxis and therapy for HIV- 1 disease. In this review, we summarize the advance in HIV-1 bNAbs discovery, maturation and further describe emerging applications in vaccine design.

A cohesive analysis of DNA/RNA sequences via entropy, energetics and spectral-domain methods to assess genomic features across single viral diversity.

In virology context, a particular virus may prevail in different forms of serotypes (as in the case of dengue 1-4 viral strains) with common and distinct genomic features. Finding such genomic details of a serogroup is useful in knowing related information for unique vaccine designs compatible for immunity across the viral diversity. For robust comparison of genomes of serovars of a virus in order to decide on their common and differential genomic details, proposed here is a set of sequence analyses exercised side-by-side via entropy, energetic and spectral-domain methods. Results obtained thereof with dengue viral serotypes, namely DEN1, DEN2, DEN3 and DEN4, are presented. Hence, inferences on distinct as well as common features extracted are annotated and indicated for possible vaccine design applications.

The Bacterial Biofilm Matrix as a Platform for Protein Delivery

ABSTRACTSurface-associated bacterial structures known as biofilms are the target of intense antimicrobial research efforts. We recently identified several secreted proteins that are retained in the bacterial biofilm matrix by their association with the biofilm exopolysaccharide scaffold. Based on our findings, we hypothesized that these problematic bacterial structures might be reengineered to serve as reservoirs for surface-active secreted proteins of biomedical, bioengineering, or biotechnological importance. By piggybacking onto one of these scaffold-associated proteins, we were able to sequester a functional enzyme to the biofilm matrix. We hypothesize that this technology may have diverse applications in vaccine design, digestive disease, and bioremediation.

Epitope Insertion at the N-Terminal Molecular Switch of the Rabbit Hemorrhagic Disease Virus T=3 Capsid Protein Leads to Larger T=4 Capsids

Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design. The RHDV capsid is based on a T=3 lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP to 8.0-Å resolution by three-dimensional cryoelectron microscopy; in addition, we used San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) capsid subunit structures to establish the backbone structure of VP1 by homology modeling and flexible docking analysis. Based on the three-domain VP1 model, several insertion mutants were designed to validate the VP1 pseudoatomic model, and foreign epitopes were placed at the N- or C-terminal end, as well as in an exposed loop on the capsid surface. We selected a set of T and B cell epitopes of various lengths derived from viral and eukaryotic origins. Structural analysis of these chimeric capsids further validates the VP1 model to design new chimeras. Whereas most insertions are well tolerated, VP1 with an FCV capsid protein-neutralizing epitope at the N terminus assembled into mixtures of T=3 and larger T=4 capsids. The calicivirus capsid protein, and perhaps that of many other viruses, thus can encode polymorphism modulators that are not anticipated from the plane sequence, with important implications for understanding virus assembly and evolution.

MultiRTA: A simple yet reliable method for predicting peptide binding affinities for multiple class II MHC allotypes

BackgroundThe binding of peptide fragments of antigens to class II MHC is a crucial step in initiating a helper T cell immune response. The identification of such peptide epitopes has potential applications in vaccine design and in better understanding autoimmune diseases and allergies. However, comprehensive experimental determination of peptide-MHC binding affinities is infeasible due to MHC diversity and the large number of possible peptide sequences. Computational methods trained on the limited experimental binding data can address this challenge. We present the MultiRTA method, an extension of our previous single-type RTA prediction method, which allows the prediction of peptide binding affinities for multiple MHC allotypes not used to train the model. Thus predictions can be made for many MHC allotypes for which experimental binding data is unavailable.ResultsWe fit MultiRTA models for both HLA-DR and HLA-DP using large experimental binding data sets. The performance in predicting binding affinities for novel MHC allotypes, not in the training set, was tested in two different ways. First, we performed leave-one-allele-out cross-validation, in which predictions are made for one allotype using a model fit to binding data for the remaining MHC allotypes. Comparison of the HLA-DR results with those of two other prediction methods applied to the same data sets showed that MultiRTA achieved performance comparable to NetMHCIIpan and better than the earlier TEPITOPE method. We also directly tested model transferability by making leave-one-allele-out predictions for additional experimentally characterized sets of overlapping peptide epitopes binding to multiple MHC allotypes. In addition, we determined the applicability of prediction methods like MultiRTA to other MHC allotypes by examining the degree of MHC variation accounted for in the training set. An examination of predictions for the promiscuous binding CLIP peptide revealed variations in binding affinity among alleles as well as potentially distinct binding registers for HLA-DR and HLA-DP. Finally, we analyzed the optimal MultiRTA parameters to discover the most important peptide residues for promiscuous and allele-specific binding to HLA-DR and HLA-DP allotypes.ConclusionsThe MultiRTA method yields competitive performance but with a significantly simpler and physically interpretable model compared with previous prediction methods. A MultiRTA prediction webserver is available at http://bordnerlab.org/MultiRTA.

Phage display and its application in vaccine design

This review focuses on phage display and its application in vaccine design. Four kinds of phage display systems and their characteristics are highlighted. Whole phage particles can be used to deliver vaccines by fusing immunogenic peptides to modified coat proteins (phage-display vaccination), or by incorporating a eukaryotic promoter-driven vaccine gene within the phage genome (phage DNA vaccination). Hybrid phage vaccination results from a combination of phage-display and phage DNA vaccination strategies, indicating the potential for evolution of phage vaccines. Phage vaccines could provide a key to unlock new approaches in combating bacterial and viral pathogens, and cancer diseases. New phage display systems will certainly emerge because of the global abundance of phage and our increasing ability to exploit them. The scope of phage display applications will continue to expand. Over the ensuing period, research should be directed toward a better understanding of the immunization mechanisms involved in phage-mediated immunization, and the development of hybrid phage vaccination.

Prediction of the binding affinities of peptides to class II MHC using a regularized thermodynamic model

BackgroundThe binding of peptide fragments of extracellular peptides to class II MHC is a crucial event in the adaptive immune response. Each MHC allotype generally binds a distinct subset of peptides and the enormous number of possible peptide epitopes prevents their complete experimental characterization. Computational methods can utilize the limited experimental data to predict the binding affinities of peptides to class II MHC.ResultsWe have developed the Regularized Thermodynamic Average, or RTA, method for predicting the affinities of peptides binding to class II MHC. RTA accounts for all possible peptide binding conformations using a thermodynamic average and includes a parameter constraint for regularization to improve accuracy on novel data. RTA was shown to achieve higher accuracy, as measured by AUC, than SMM-align on the same data for all 17 MHC allotypes examined. RTA also gave the highest accuracy on all but three allotypes when compared with results from 9 different prediction methods applied to the same data. In addition, the method correctly predicted the peptide binding register of 17 out of 18 peptide-MHC complexes. Finally, we found that suboptimal peptide binding registers, which are often ignored in other prediction methods, made significant contributions of at least 50% of the total binding energy for approximately 20% of the peptides.ConclusionsThe RTA method accurately predicts peptide binding affinities to class II MHC and accounts for multiple peptide binding registers while reducing overfitting through regularization. The method has potential applications in vaccine design and in understanding autoimmune disorders. A web server implementing the RTA prediction method is available at http://bordnerlab.org/RTA/.

Immunopotentiating Effects of Four Chinese Herbal Polysaccharides Administered at Vaccination in Chickens

This study was conducted to evaluate the effects of 4 Chinese herbal polysaccharides on the production of serum antibodies and the proliferation of peripheral T lymphocytes, including subpopulations in vaccinated chickens. A total of 450 chickens were randomly assigned to 9 groups at 14 d of age and vaccinated first with live Newcastle disease (ND)-infectious bronchitis virus vaccine, and second with ND-infectious bronchitis oil adjuvant vaccine at 28 d of age. At the same time as the first vaccination, the chickens in groups 1 to 8 were intramuscularly injected with 4 polysaccharides at high and low dosages, respectively, once a day for 3 successive days starting on the day of the first vaccination. Group 9 (control group) was injected in the same manner with saline instead of a polysaccharide. On d 7, 14, 21, 28, 35, 42, and 49 after the first vaccination, the temporal changes in serum ND hemagglutination inhibition antibody titer were determined by the micromethod. On d 10, 20, 30, 40, and 50 after the first vaccination, the proliferation of peripheral blood mononuclear cells in response to concanavalin A stimulation as well as the proportions of CD3+, CD4+, and CD8+ peripheral blood mononuclear cells were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and flow cytometry, respectively. The results showed that astragalus polysaccharide and isatis root polysaccharide at low dosages, and achyranthes root polysaccharide and Chinese yam polysaccharide at high dosages significantly enhanced the ND antibody titers, concanavalin A-induced proliferation of peripheral blood lymphocytes, and ratio of CD4+ to CD8+ (P < 0.05). Collectively, these findings indicate that the 4 polysaccharides possess significant immune-enhancing properties in chickens. This finding may have direct application in vaccine design and other strategies designed to potentiate immune system development and function in chickens.

Intranasal immunisation with recombinant Lactococcus lactis displaying either anchored or secreted forms of Proteus mirabilis MrpA fimbrial protein confers specific immune response and induces a significant reduction of kidney bacterial colonisation in mice

Proteus mirabilis, a common cause of urinary tract infections in humans, can express different fimbriae. MR/P fimbriae may contribute to bacterial colonisation, and its structural protein MrpA represents a promising candidate antigen for mucosal vaccination. Commercial complex vaccines have limited, short-lived protection and are incapable of eliciting mucosal responses against putative antigens related to virulence. The development of mucosal live vaccines using food-grade lactic acid bacterium Lactococcus lactis as antigen vehicle is an attractive alternative and a safe vaccination strategy against P. mirabilis infection. Here, we report the construction of L. lactis strains modified to produce MrpA via two cellular locations, cell wall-anchored and secreted. Protection assays against P. mirabilis infection and evaluation of the immune response generated after immunisation were conducted in a mouse model. MrpA protein was efficiently expressed by L. lactis strain and caused a significant induction of specific serum IgG and IgA in the animals immunised with L. lactis pSEC: mrpA and L. lactis pCWA: mrpA respectively. A significant reduction of renal bacterial colonisation was observed in both groups of mice ( P < 0.05) after P. mirabilis challenge. This is the first example of a P. mirabilis fimbrial antigen expressed in a food-grade live strain with promising applications in vaccine design.

Reliability of Computer Algorithm-Based Binding Predictions for the Identification of Leukemia Antigens.

Major Histocompatibility Complex class I (MHC-I) molecules present antigenic peptides to T cells on the cell surface as a prerequisite for stimulating cytotoxic T cell response. Thus, the ability to reliably identify the peptides that can bind to MHC molecules is of practical importance for rapid vaccine development. Several computer-based prediction methods have been applied to study the interaction of MHC class I/peptide binding. Here we have compared three of the most commonly used predictive algorithms BIMAS, SYFPEITHI and Rankpep with actual binding of HLA-A*0201 peptides in vitro. Forty six HLA-A*0201 peptides were selected from several target oncoproteins: Wilms' tumor (WT1), native and imatinib- mutated bcr-abl p210 and JAK2 protein. Experimental peptide binding to HLA-A*0201 was assessed using a MHC stabilization assay on T2, TAP deficient cells. Peptides were considered to show positive in vitro binding if the mean fluorescence was at least 50 % of the binding of a high affinity reference peptide. Peptides qualified as positive in vitro if the BIMAS score was ≥ 100, the SYFPEITHI score ranked ≥ 24 or the Rankpep was ≥ 50. Results are summarized below:BIMASSYFPEITHIRANKPEPSensitivity84 %72 %60 %Specificity76 %71 %81 %Positive Predictive Value84 %72 %60 %Negative Predictive Value80 %68 %63 %Combining two or more computer methods did not appear to improve the predictive value. In conclusion, of the three predictive algorithms, the best correspondence with the actual MHC binding was demonstrated with the BIMAS algorithm. Predictive computer algorithms are important for preselection of potential T-cell epitope candidates for the application in vaccine design.

Carbohydrate Mimotopes in the Rational Design of Cancer Vaccines

The task of rationally designing vaccines that can effectively impact on the survival of cancer patients remains challenging. Monoclonal antibodies and T cell receptors have proven to be viable templates for the application of pharmacophore design principles to develop antigens and immunogens as these immune system molecules recognize a variety of sequentially and structurally unrelated ligands. This structural information combined with immunological assessment has contributed to the development of strategies to elicit effective humoral and cellular responses to cancer cells. Understanding the structural requirements for antibody and T cell recognition provides a basis for identifying potentially new sets of immunogens that may have both fundamental immunological and clinical value. Here we review the structural concepts and approaches used in vaccine design applications that illustrate the value and limitations of using chemical (peptide libraries) and immunological information to define novel peptide immunogens that function as mimotopes to generate immune responses targeting tumor associated carbohydrate antigens.

Display and release of the Plasmodium falciparum circumsporozoite protein using the autotransporter MisL of Salmonella enterica

The Salmonella enterica MisL (protein of membrane insertion and secretion) is an autotransporter with high homology to AIDA-I (adhesin involved in diffuse adherence) of enteropathogenic Escherichia coli. Considering that it has been reported that the MisL β translocator domain is able to display heterologous passenger peptides to the bacterial surface, we developed a system to display proteins and release them to the external environment by means of proteolytic cleavage. Plasmids were constructed encoding 8 or 53 repeats of the NANP (Asp–Ala–Asp–Pro) tetrapeptide, which is the main B cell epitope of the Plasmodium falciparum circumsporozoitic protein (CSP), fused to the the MisL β-domain and including the recognition cleavage sequence from the E. coli OmpT surface protease. E. coli XL-10Gold and BL21(DE3) (OmpT positive and negative, respectively) and Salmonella enterica serovar Typhimurium SL3261 (Aro A −) were transformed with the plasmids and, both expression and localization of the fusion proteins were assessed by Western blot, indirect immunofluorescence, and flow cytometry, using a monoclonal antibody against (NANP) 3. Higher expression of the (NANP) 8 and (NANP) 53 fusion proteins was demonstrated on the bacterial surface of the OmpT negative E. coli strains and the (NANP) 53 in the culture supernatant of E. coli XL-10Gold indicating a protease mediated cleavage. The flow cytometry analysis suggested 71 and 98% cleavage efficiency for the (NANP) 8 and (NANP) 53, respectively, in E. coli XL-10Gold. Similar results were obtained in S. enterica serovar Typhimurium SL3261, suggesting the involvement of other proteases related to OmpT. These results demonstrate that MisL may be used for the autodisplay and release of passenger proteins in attenuated Salmonella or E. coli strains, which may have several applications in vaccine design.

Directing the Immune Response to Carbohydrate Antigens

Peptide mimetics may substitute for carbohydrate antigens in vaccine design applications. At present, the structural and immunological aspects of antigenic mimicry, which translate into immunologic mimicry, as well as the functional correlates of each, are unknown. In contrast to screening peptide display libraries, we demonstrate the feasibility of a structure-assisted vaccine design approach to identify functional mimeotopes. By using concanavalin A (ConA), as a recognition template, peptide mimetics reactive with ConA were identified. Designed peptides were observed to compete with synthetic carbohydrate probes for ConA binding, as demonstrated by enzyme-linked immunosorbent assay and isothermal titration calorimetry (ITC) analysis. ITC measurements indicate that a multivalent form of one particular mimetic binds to ConA with similar affinity as does trimannoside. Splenocytes from mimeotope-immunized mice display a peptide-specific cellular response, confirming a T-cell-dependent nature for the mimetic. As ConA binds to the Envelope protein of the human immunodeficiency virus, type 1 (HIV-1), we observed that mimeotope-induced serum also binds to HIV-1-infected cells, as assessed by flow cytometry, and could neutralize T-cell line adapted HIV-1 isolates in vitro, albeit at low titers. These studies emphasize that mimicry is based more upon functional rather than structural determinants that regulate mimeotope-induced T-dependent antibody responses to polysaccharide and emphasize that rational approaches can be employed to develop further vaccine candidates.

Design and immunological properties of topographic immunogenic determinants of a protein antigen (LDH-C4) as vaccines.

Antibodies elicited by immunization with short peptides containing antigenic determinants have been shown, in general, to bind with greatly reduced affinity to the corresponding region in the native proteins. Thus, contiguous linear peptides have not proven to be effective immunogens in generating high affinity neutralizing or protective antibodies and consequently appear to be poor prospects for vaccines. The molecular basis for such reduced reactivity is clear from the crystal structure determination of antibody Fabs bound to protein antigens, which showed the complementarity between interfaces to be lock-and-key-like and extending over a large area (750 A2) involving discontinuous segments of the polypeptide chain. Thus, small perturbations in the secondary and tertiary structure of the antigen have profound effects on the fit of the antigen and its corresponding antibody. Because short peptides are unlikely to assume any particular conformation in solution, the fit is likely to be poor. New strategies are therefore required to produce conformationally stable peptides that mimic the critical structural features of the protein antigenic site. Here we show that a putative topographic determinant of the testis-specific isozyme of lactate dehydrogenase C4 (LDH-C4), designed and synthesized to adopt a well defined alpha-helical secondary and tertiary structure (four-helix bundle motif) in aqueous solutions, is highly immunogenic in both rabbits and mice, inducing IgG antibodies that bind to native LDH-C4. This engineered conformational 40-residue peptide is considerably more effective in inducing antibodies, as compared with the corresponding linear peptide. The antibody response is obtained without coupling the peptide to a carrier protein, suggesting that the peptide contains a T-cell antigenic determinant. The strategy described here to produce a conformationally stable peptide that mimics the native structure may have general applications in vaccine design.