Synthetic peptides in the diagnosis of HIV infection

Peptide-based enzyme-linked immunosorbent assays have been found to be enough sensitive and specific for the diagnosis of human immunodeficiency virus specific antibodies in acquired immunodeficiency syndrome patients. This review provides an overview of the most important peptides developed for use as synthetic antigens in immunodiagnosis of HIV-infected patients. In particular, many studies have been devoted to discriminate between the two retroviruses HIV-1 and HIV-2, as well as different subtypes.     

Immunodiagnosis of parasitic diseases with synthetic peptides

Parasitic diseases remain as a major public health problem worldwide, not only based on their historically high morbidity and mortality rates, but also because risk factors associated with their transmission are increasing. Laboratory diagnosis and particularly immunodiagnosis is a basic tool for the demonstration, clinical management and control of these infections. Classically, the serological tests for the detection of antibodies or antigens are based on the use of crude and purified antigens. Synthetic peptides have opened a new field and perspectives, as the source of pure epitopes and molecules for diagnosis of malaria, Chagas' disease, leishmaniasis, schistosomiasis, hidatidosis, cysticercosis and fasciolosis based on the detection of antibodies and circulating antigens. Herein, are critically reviewed the relevant advances and applications of the synthetic peptides on immunodiagnosis of parasitic diseases. A variety of sequences, constructs (monomers, polymers, MAPs), immunological methods and samples have been used, demonstrating their diagnostic potential. However, in most parasitic infections it is necessary to use more than a single peptide in order to avoid the genetic restriction against certain epitopes, as well as to test them in well characteized groups of patients, in order to confirm their sensitivity and specificity. The concept of multidiagnosis with synthetic peptides, using a novel multi-dot blot assay is introduced. Finally, the chemical imitation of antigens, offers a tremendous posibilities in the diagnosis of parasitic infections in developing countries since this strategy is cheaper, simpler, reproducible, useful for large scale testing and in most cases, specific and sensitive.     

From the Laboratory to the Clinic: A Retrospective on Fully Synthetic Carbohydrate-Based Anticancer Vaccines Frequently used abbreviations are listed in the appendix

This review provides an account of our explorations into oligosaccharide and glycoconjugate construction for the creation and evaluation of vaccines based on carbohydrate-centered tumor antigens. Our starting point was the known tendency of transformed cells to express selective carbohydrate motifs in the form of glycoproteins or glycolipids. Anticancer vaccines derived from carbohydrate-based antigens could be effective targets for immune recognition and attack. Obtaining significant quantities of such structures from natural sources is, however, extremely difficult. With the total synthesis of tumor-associated carbohydrate antigens accomplished, we began to evaluate at the clinical level whether the human immune system can respond to such fully synthetic antigens in a focused and useful way. Toward this goal, we have merged the resources of chemistry and immunology in an attack on the problem. The synthesis and immunoconjugation of various tumor-associated carbohydrate antigens and the results of such constructs in mice vaccinations will be described. For fashioning an effective vaccine, conjugation to a suitable immunogenic carrier was necessary and conjugates of KLH (keyhole limpet cyanin) have consistently demonstrated the relevant immunogenicity. Preclinical and clinical studies with synthetic conjugate carbohydrate vaccines show induction of IgM- and IgG-antibody responses. Another approach to anticancer vaccines involves the use of clustered glycopeptides as targets for immune attack. Initial attention has been directed to mucin related O-linked glycopeptides. Synthetic trimeric clusters of glycoepitopes derived from the Tn-, TF- and Lewis(y)-antigens, appropriately bioconjugated, have been demonstrated to be immunogenic. The hope is that patients immunized in an adjuvant manner with synthetic carbohydrate vaccines would produce antibodies reactive with cancer cells and that the production of such antibodies would mitigate against tumor spread, thereby enabling a more favorable survival and "quality of life" prognosis.     

Design of synthetic peptides for diagnostics

Due to the advantageous properties of synthetic molecules compared to biological ones biological molecules in diagnostic tests are replaced increasingly by synthetic ones, usually synthetic peptides or related molecules. The replacement of biological antigens by synthetic peptides is most advanced at present, as well as the use of site-specific antibodies induced with synthetic peptides. Moreover recent results indicate that synthetic molecules may also replace antibodies. Ultimately this will lead to diagnostic assays built of synthetic molecules only.     

合成多肽疫苗研究进展

综述了合成多肽疫苗的研究进展,系统地介绍了目前寻找抗原肽的各种方法以及各类可用作疫苗接种的合成多肽抗原的制备和应用.     

Branched peptides as therapeutics

The concept of 'magic bullet', initially ascribed to immunoglobulins by Paul Ehrlich at the beginning of the 20th century and strengthened by the hybridoma technology of Kohler and Milstein in the mid 70s, can nowadays be attributed to different target-specific molecules, such as peptides. This attribution is increasingly valid in light of the explosion of new technologies for peptide library construction and screening, not to mention improvements in peptide synthesis and conjugation and in-vivo peptide stability, which make peptide molecules specific bullets for targeting pathological markers and pathogens. Today, hundreds of peptides are being developed and dozens are in clinical trials for a variety of diseases, demonstrating that the general reluctance towards peptide drugs that existed a decade ago has now been overcome. In spite of this progress, the development of new peptide drugs has largely been limited by their short half-life. Branched peptides such as Multiple Antigen Peptides (MAPs) were invented in the 80s by Tam [Tam, J.P., (1998) Proc. Natl. Acad. Sci. USA, 85, 5409] and have been extensively tested to reproduce single epitopes to stimulate the immune system for new vaccine discovery. In our lab we discovered that MAP molecules acquire strong resistance to proteases and peptidases. This resistance renders MAPs very stable and thus suitable for drug development. Here we report our experience with several MAP molecules in different biotechnological applications ranging from antimicrobial and anti toxin peptides to peptides for tumor targeting.     

Optimized sample preparation for MALDI mass spectrometry analysis of protected synthetic peptides

The recent development and commercialization of Fuzeon (enfuvirtide) demonstrated that a convergent strategy comprised of both solid- and solution-phase synthetic methodologies presents a viable route for peptide manufacturing on a multi-ton scale. In this strategy, the target sequence is prepared by stepwise solid-phase synthesis of protected peptide fragments, which are then coupled together in the solution-phase to give the full-length sequence. These synthetic methodologies pose a unique challenge for mass spectrometry (MS), as protected peptide intermediates are often marked by poor solubility, structural lability, and low ionization potential. Matrix-assisted laser desorption/ionization (MALDI) MS is uniquely suited to such analytes; however, generalized protocols for MALDI analysis of protected peptides have yet to be demonstrated. Herein, we report an operationally simple sample preparation method for MALDI analysis of protected peptides, which greatly facilitates the collection and interpretation of MS data. In this method, the difficulty in MS analysis of protected peptides has been greatly diminished by use of dithranol as a matrix and CsCl as an additive, giving rise to intentionally-formed Cs(+) adducts. With greatly reduced fragmentation, better crystalline morphology, and easier data interpretation, we anticipate that these findings will find utility in peptide process development and manufacturing settings for reaction monitoring, troubleshooting, and quality control.     

A mini-review and perspective on multicyclic peptide mimics of antibodies

This review gave a brief introduction on recent development in monocyclic and multicyclic peptide mimics of antibodies and provides a perspective on screening and design of multicyclic peptide mimics of antibodies in the future.     

Rapid Photolysis-Mediated Folding of Disulfide-Rich Peptides

Structure–activity relationship studies are a highly time-consuming aspect of peptide-based drug development, particularly in the assembly of disulfide-rich peptides, which often requires multiple synthetic steps and purifications. Therefore, it is vital to develop rapid and efficient chemical methods to readily access the desired peptides. We have developed a photolysis-mediated “one-pot” strategy for regioselective disulfide bond formation. The new pairing system utilises two ortho-nitroveratryl protected cysteines to generate two disulfide bridges in less than one hour in good yield. This strategy was applied to the synthesis of complex disulfide-rich peptides such as Rho-conotoxin ρ-TIA and native human insulin.     

GB virus C (GBV-C) / hepatitis G virus (HGV): towards the design of synthetic peptides-based biosensors for immunodiagnosis of GBV-C/HGV infection

In the present study, new putative epitopes located in structural (E2) and non-structural (NS3) proteins of GBV-C/HGV were identified by computer-aided prediction of antigenicity and synthesized in solid-phase, following an Fmoc/tBut strategy, for their use in immunoassays. The corresponding synthetic peptides were used as antigens in ELISA assays and in real-time biospecific interaction measurements. This last approach allowed direct detection of GBV-C/HGV-specific antibodies in human sera. Good correlations were obtained between the biospecific interaction analysis and the ELISA. To verify the performance of these new assays in comparison to the existing recombinant E2 protein commercial test, antibodies to synthetic peptides were searched for in different panels of serum samples. The main conclusion of this work is the usefulness of E2 peptides in the detection of antibodies. Moreover, the NS3 peptide could be exploited to improve the sensitivity of the currently available test. Our results offer a new approach to develop new diagnostic peptide based biosensors for serodiagnosis of GBV-C/HGV infection.     

Antibodies to short synthetic peptides for specific recognition of partly denatured protein

On immunization with subclasses of homologous proteins, the presence of immunodominant class-specific structures often prevents the development of subclass specific antibodies. Immunization with synthetic peptides representing specific structures in such a protein can then be used instead. Sequences derived from human IgG2 and Staphylococcus aureus enterotoxin were used as a model to develop general criteria for immunization with synthetic peptides. Peptide sequences were selected on the basis of criteria such as hydrophilic property, surface probability and secondary structure, which together constitute an antigenic index. The carrier proteins keyhole limpet hemocyanin and chicken gammaglobulin were coupled by using either carbodiimide or a maleimide. Peptide-specific responses can be elicited regardless of the sequence. Some coupling conditions influenced the specificity of the antibody responses. The results of immunization with short peptides not representing a natural B-cell determinant of a protein suggest that the frequency of native protein-recognizing antibodies is considerably lower than that of antibodies that recognize the protein in a partly denatured or altered state.     

Multiple Peptide Synthesis Methods and Their Applications. New Synthetic Methods (87)

Rapid developments in the biotechnology of new proteins, as well as advances in immunology and the development of pharmaceuticals based on inhibitors and antagonists, have led to immense demands for synthetic peptides. Simultaneous preparation of 100–150 completely different peptides, having chain lengths of up to 20 amino acids can nowadays be achieved using multiple synthesis methods. The yields and qualities of the peptides so obtained are high enough to permit reliable in vivo and in vitro screening for biological activities. Moreover, it is possible to optimize synthetic conditions and to carry out comparative studies on the secondary structures and conformational mapping of proteins. Special multiple synthesis methods facilitate the epitope mapping of larger peptides for diagnostic purposes and for the development of vaccines based on a few hundreds of free or rod-bound peptides that are useful for immunoassays. Multiple methods of peptide synthesis also enable the preparation of so-called peptide libraries which could comprise hundreds of thousands of peptides, and by which new perspectives for the screening of lead structures will be opened up. Peptide synthesis using a combination of photolabile protecting groups and photolithographic procedures enables the assembling of peptide libraries on small plates for use in miniature immunoassays. Furthermore, lipopeptide-antigen conjugates allow both the preparation of peptide-specific and monoclonal antibodies as well as a complete screening of epitopes of B-, T-helper and T-killer cells. Applications in the areas of AIDS diagnosis, the development of vaccines, and screening for the hormone analogues, demonstrate just some of the possibilities that have been opened up by multiple peptide synthesis methods.     

Synthetic peptides in the diagnosis of systemic autoimmune diseases

Proteins recognized by antibodies from patients with autoimmune diseases have been intensively studied over the two past decades since cDNAs encoding autoantigens have become available. Identity of many of them has been defined, and specific structural motifs or post-translational modifications, which may be important to explain the generation of such antibodies during the autoimmune process, have been pointed out. Immunological analysis of sera from autoimmune patients with recombinant fragments and with short peptides has revealed the presence of dominant epitopes along proteins; some of them are targeted by antibodies from patients with specific diseases or disease subsets. Innovative technologies such as peptide arrays and biosensors as well as the exploitation of large peptides libraries have recently open up new perspectives. Peptides bearing natural modifications, peptide analogues, as well as mimotopes of protein or non-protein antigens (DNA, RNA, sugar) have been developed and might advantageously replace native antigens in routine immunoassays. Although numerous conformational epitopes have not yet been identified, and cannot be identified by the approaches classically used in epitope mapping studies, such peptides and peptide analogues may represent efficient probes to detect the presence of circulating autoantibodies in the serum of autoimmune patients and help for establishing specific and sensitive early diagnostic tests. They may also lead to the design of high-affinity ligands for purifying autoantibodies. These different aspects are discussed and epitope mapping studies of a number of autoantigens (e.g. histones, sn and hnRNP proteins and Ro proteins) are summarized.     

Different approaches to potentiate the immune response induced by a 12-mer synthetic peptide

Interest in synthetic immunogenic peptides is increasingly growing due to the continuous achievements in the understanding of the molecular basis of the immune response. Moreover, the use of peptide fragments to generate antibodies capable of recognizing and neutralizing viral particles could be a valuable alternative to conventional vaccines: they are safe, they can be designed to induce defined immune responses and they can be synthesised in large quantities in high purity. However, their relatively low immunogenicity requires the development of effective adjuvants and/or their incorporation into controlled release formulations. Several different strategies have been used for the induction and analysis of protective immune responses induced by short peptides against infectious diseases. In the present article we summarise the different approaches used to potentiate the immune response induced by a continuous epitope of hepatitis A virus: co-linear link of T-cell epitopes in different orientations, incorporation into liposomes, and preparation of homogeneous and heterogeneous Multiple Antigenic Peptides.     

Synthetic peptides in the diagnosis of neurological diseases

For several years peptides have been used to investigate many aspects of the molecular mechanisms supporting the etiopathogenesis of neurological diseases. Even if well-established and commercially-available assays for the diagnosis of neurological diseases, based on peptides, are presently not available, much research has been focused to the comprehension of the pathogenetic mechanisms of relevant neurological diseases, such as Multiple Sclerosis, Alzheimer's disease and prion diseases. Several peptides, which strongly contributed to increase in the progress, and in the understanding of the molecular mechanisms of these important diseases, have been selected as antigens to be proposed in possible alternative diagnostic methodologies. The authors focused their attention on the possible peptides to be used as diagnostics for these three characteristic neurological diseases. In fact, Multiple Sclerosis is an autoimmune disease in which peptides have been used for the characterization of both B and T immune response, while Alzheimer's disease and prion diseases can be cumulative regarded as "protein folding disorders" in which peptides can be used to identify the misfolded protein.     

Designing anti-Zika virus peptides derived from predicted human-Zika virus protein-protein interactions

The production of anti-Zika virus (ZIKV) therapeutics has become increasingly important as the propagation of the devastating virus continues largely unchecked. Notably, a causal relationship between ZIKV infection and neurodevelopmental abnormalities has been widely reported, yet a specific mechanism underlying impaired neurological development has not been identified. Here, we report on the design of several synthetic competitive inhibitory peptides against key pathogenic ZIKV proteins through the prediction of protein–protein interactions (PPIs). Often, PPIs between host and viral proteins are crucial for infection and pathogenesis, making them attractive targets for therapeutics. Using two complementary sequence-based PPI prediction tools, we first produced a comprehensive map of predicted human-ZIKV PPIs (involving 209 human protein candidates). We then designed several peptides intended to disrupt the corresponding host-pathogen interactions thereby acting as anti-ZIKV therapeutics. The data generated in this study constitute a foundational resource to aid in the multi-disciplinary effort to combat ZIKV infection, including the design of additional synthetic proteins.     

Rapid colorimetric detection of antibody-epitope recognition at a biomimetic membrane interface.

Biomolecular recognition of antigens and epitopes by antibodies is a fundamental event in the initiation of immune response and plays a central role in a variety of biochemical processes. Peptide binding requires, in many cases, presentation of the peptides at interfaces, such as protein surfaces, cellular membranes, and synthetic polymer surfaces. We describe a novel molecular system in which interactions between antibodies and peptide epitopes displayed at a biomimetic membrane interface can be detected through induction of visible, rapid color transitions. The colorimetric assembly consists of a phospholipid/polydiacetylene matrix anchoring a hydrophobic peptide displaying the epitope at its N-terminus. The colorimetric transitions observed in the assembly, corresponding to perturbation of the polydiacetylene framework, are induced only upon recognition of the displayed epitope by its specific antibody present in the aqueous solution. Significantly, the color changes occur after a single mixing step, without further chemical reactions or enzymatic processing. The new molecular system could be utilized for studying antigen-antibody interactions and peptide-protein recognition, epitope mapping, and rapid screening of biological and chemical libraries.     

Synthetic Methods of Phosphonopeptides

Phosphonopeptides are phosphorus analogues of peptides and have been widely applied as enzyme inhibitors and antigens to induce catalytic antibodies. Phosphonopeptides generally contain one aminoalkylphosphonic acid residue and include phosphonopeptides with C-terminal aminoalkylphosphonic acids and phosphonopeptides with a phosphonamidate bond. The phosphonamidate bond in the phosphonopeptides is generally formed via phosphonylation with phosphonochloridates, condensation with coupling reagents and enzymes, and phosphinylation followed by oxidation. Pseudo four-component condensation reaction of amides, aldehydes, alkyl dichlorophosphites, and amino/peptide esters is an alternative, convergent, and efficient strategy for synthesis of phosphonopeptides through simultaneous construction of aminoalkylphosphonic acids and formation of the phosphonamidate bond. This review focuses on the synthetic methods of phosphonopeptides containing a phosphonamidate bond.