In Silico-Guided Sequence Modification of Epitopes in Cancer Vaccine Development.

Discovery of tumor antigenic epitopes is important for cancer vaccine development. Such epitopes can be designed and modified to become more antigenic and immunogenic in order to overcome immunosuppression towards the native tumor antigen. In silico-guided modification of epitope sequences allows predictive discrimination of those that may be potentially immunogenic. Therefore, only candidates predicted with high antigenicity will be selected, constructed, and tested in the lab. Here, we described the employment of in silico tools using a multiparametric approach to assess both potential T-cell epitopes (MHC class I/II binding) and B-cell epitopes (hydrophilicity, surface accessibility, antigenicity, and linear epitope). A scoring and ranking system based on these parameters was developed to shortlist potential mimotope candidates for further development as peptide cancer vaccines.

A Coreceptor-Mimetic Peptide Enhances the Potency of V3-Glycan Antibodies.

Broadly neutralizing antibodies (bNAbs) target five major epitopes on the HIV-1 envelope glycoprotein (Env). The most potent bNAbs have median half-maximal inhibitory concentration (IC50) values in the nanomolar range, and the broadest bNAbs neutralize up to 98% of HIV-1 strains. The engineered HIV-1 entry inhibitor eCD4-Ig has greater breadth than bNAbs and similar potency. eCD4-Ig is markedly more potent than CD4-Ig due to its C-terminal coreceptor-mimetic peptide. Here we investigated whether the coreceptor-mimetic peptide mim6 improved the potency of bNAbs with different epitopes. We observed that when mim6 was appended to the C terminus of the heavy chains of bNAbs, this sulfopeptide improved the potency of all classes of bNAbs against HIV-1 isolates that are sensitive to neutralization by the sulfopeptide alone. However, mim6 did not significantly enhance neutralization of other isolates when appended to most classes of bNAbs, with one exception. Specifically, mim6 improved the potency of bNAbs of the V3-glycan class, including PGT121, PGT122, PGT128, and 10-1074, by an average of 2-fold for all HIV-1 isolates assayed. Despite this difference, 10-1074 does not induce exposure of the coreceptor-binding site, and addition of mim6 to 10-1074 did not promote shedding of the gp120 subunit of Env. Mixtures of 10-1074 and an Fc domain fused to mim6 neutralized less efficiently than a 10-1074/mim6 fusion, indicating that mim6 enhances the avidity of this fusion. Our data show that mim6 can consistently improve the potency of V3-glycan antibodies and suggest that these antibodies bind in an orientation that facilitates mim6 association with Env.IMPORTANCE HIV-1 requires both the cellular receptor CD4 and a tyrosine-sulfated coreceptor to infect its target cells. CD4-Ig is a fusion of the HIV-1-binding domains of CD4 with an antibody Fc domain. Previous studies have demonstrated that the potency of CD4-Ig is markedly increased by appending a coreceptor-mimetic sulfopeptide to its C terminus. We investigated whether this coreceptor-mimetic peptide improves the potency of broadly neutralizing antibodies (bNAbs) targeting five major epitopes on the HIV-1 envelope glycoprotein (Env). We observed that inclusion of the sulfopeptide dramatically improved the potency of all bNAb classes against isolates with more-open Env structures, typically those that utilize the coreceptor CXCR4. In contrast, the sulfopeptide improved only V3-glycan antibodies when neutralizing primary isolates, on average by 2-fold. These studies improve the potency of one class of bNAbs, show that coreceptor-mimetic sulfopeptides enhance neutralization through distinct mechanisms, and provide insight for the design of novel multispecific entry inhibitors.

Design, Synthesis, and Immunological Evaluation of a Multicomponent Construct Based on a Glycotripeptoid Core Comprising B and T Cell Epitopes and a Toll-like Receptor 7 Agonist That Elicits Potent Immune Responses.

We present here for the first time the synthesis and immunological evaluation of a fully synthetic three-component anticancer vaccine candidate that consists of a ß-glycotripeptoid core mimicking a cluster of Tn at the surface of tumor cells (B epitope), conjugated to the OVA 323-339 peptide (T-cell epitope) and a Toll-like receptor 7 (TLR7) agonist for potent adjuvanticity. The immunological evaluation of this construct and of precursor components demonstrated the synergistic activity of the components within the conjugate to stimulate innate and adaptive immune cells (DCs, T-helper, and B-cells). Surprisingly, immunization of mice with the tricomponent GalNAc-based construct elicited a low level of anti-Tn IgG but elicited a very high level of antibodies that recognize the TLR7 agonist. This finding could represent a potential vaccine therapeutic approach for the treatment of some autoimmune diseases such as lupus.

Recombinant Peptidomimetic-Nano Luciferase Tracers for Sensitive Single-Step Immunodetection of Small Molecules.

Phage borne peptides isolated from phage libraries have proven to be valuable reagents for the development of small-molecule immunoassays. However, the large size, low diffusion rate, and biological nature of the phage particles create some limitations to their use and require secondary reagents for its detection. In this work, we explore the use of the Nano luciferase (NanoLuc) as a fusion partner to generate recombinant tracers for immunoassay development. The imidaclothiz peptidomimetic C2-15 that specifically binds to the anti-imidaclothiz monoclonal antibody (mAb) 1E7 was fused to NanoLuc, both at the N terminus (C2-15-NanoLuc) and C terminus (NanoLuc-C2-15). NanoLuc-C2-15 showed better performance than C2-15-NanoLuc and was adopted to develop a bioluminescent enzyme immunoassay (BLEIA) and a bioluminescence lateral flow immunoassay (BLLFIA) for imidaclothiz. The luminescence signal of NanoLuc-C2-15 rapidly reaches high intensity with slow attenuation, which enabled one to capture the BLLFIA readout by using a smartphone without an external light source. The IC50 of the BLEIA and BLLFIA were 3.3 ± 0.2 and 6.4 ± 0.4 ng mL-1, respectively. Both immunoassays exhibited good accuracy for the detection of imidaclothiz in environmental and agricultural samples.

Peptide mimetics of immunoglobulin A (IgA) and FcαRI block IgA-induced human neutrophil activation and migration.

The cross-linking of the IgA Fc receptor (FcαRI) by IgA induces release of the chemoattractant LTB4, thereby recruiting neutrophils in a positive feedback loop. IgA autoantibodies of patients with autoimmune blistering skin diseases therefore induce massive recruitment of neutrophils, resulting in severe tissue damage. To interfere with neutrophil mobilization and reduce disease morbidity, we developed a panel of specific peptides mimicking either IgA or FcαRI sequences. CLIPS technology was used to stabilize three-dimensional structures and to increase peptides' half-life. IgA and FcαRI peptides reduced phagocytosis of IgA-coated beads, as well as IgA-induced ROS production and neutrophil migration in in vitro and ex vivo (human skin) experiments. Since topical application would be the preferential route of administration, Cetomacrogol cream containing an IgA CLIPS peptide was developed. In the presence of a skin permeation enhancer, peptides in this cream were shown to penetrate the skin, while not diffusing systemically. Finally, epitope mapping was used to discover sequences important for binding between IgA and FcαRI. In conclusion, a cream containing IgA or FcαRI peptide mimetics, which block IgA-induced neutrophil activation and migration in the skin may have therapeutic potential for patients with IgA-mediated blistering skin diseases.

Development of lipopolysaccharide-mimicking peptides and their immunoprotectivity against Vibrio cholerae serogroup O1.

Vibrio cholerae serogroup O1 is the main causative agent of cholera diseases defined by life threatening rice watery diarrhea. Cholera routine vaccination has failed in controlling epidemics in developing countries because of their hard and expensive production. In this study, our aim was to investigate phage displayed mimotopes that could mimic V. cholerae lipopolysaccharide (LPS). Although LPS of Vibrio, as an endotoxin, can stimulate the immune system, thereby making it a suitable candidate for cholera vaccine, its toxicity remains as a main problem. Phage particles displaying 12 amino acid peptides were selected from phage library mimicking the antigenic epitopes of LPS from vibrio. The screening was carried out using single-domain antibody fragment VHH against LPS as target through three rounds of selection. Three clones with highest affinity to VHH were selected. To find out a new and efficient vaccine against cholera, these three phage particles containing high-affinity peptides were administered to mice to investigate the active and passive immunity. Out of 20 particles, three showed the highest affinity toward VHH. ELISA was carried out with immunized mice sera using LPS and three selected phages particles individually. ETEC, Shigella sonnei, and clinical isolates were used as bacterial targets. These three selected phages (individually or in combination) could stimulate mice immune system producing active and passive immunity. The mice immunized with phage particles could protect about 14 LD50 of V. cholerae. In conclusion, these peptides are mimicking LPS and can potentially act as vaccine candidates against V. cholerae. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Identification of Peptide Mimics of a Glycan Epitope on the Surface of Parasitic Nematode Larvae.

Phage display was used to identify peptide mimics of an immunologically protective nematode glycan (CarLA) by screening a constrained C7C peptide library for ligands that bound to an anti-CarLA mAb (PAB1). Characterisation of these peptide mimotopes revealed functional similarities with an epitope that is defined by PAB1. Mimotope vaccinations of mice with three selected individual phage clones facilitated the induction of antibody responses that recognised the purified, native CarLA molecule which was obtained from Trichostrongylus colubriformis. Furthermore, these mimotopes are specifically recognised by antibodies in the saliva of animals that were immune to natural polygeneric nematode challenge. This shows that antibodies to the PAB1 epitope form part of the mucosal polyclonal anti-CarLA antibody response of nematode immune host animals. This demonstrates that the selected peptide mimotopes are of biological relevance. These peptides are the first to mimic the PAB1 epitope of CarLA, a defined larval glycan epitope which is conserved between many nematode species.

Unsupervised HLA Peptidome Deconvolution Improves Ligand Prediction Accuracy and Predicts Cooperative Effects in Peptide-HLA Interactions.

Ag presentation on HLA molecules plays a central role in infectious diseases and tumor immunology. To date, large-scale identification of (neo-)Ags from DNA sequencing data has mainly relied on predictions. In parallel, mass spectrometry analysis of HLA peptidome is increasingly performed to directly detect peptides presented on HLA molecules. In this study, we use a novel unsupervised approach to assign mass spectrometry-based HLA peptidomics data to their cognate HLA molecules. We show that incorporation of deconvoluted HLA peptidomics data in ligand prediction algorithms can improve their accuracy for HLA alleles with few ligands in existing databases. The results of our computational analysis of large datasets of naturally processed HLA peptides, together with experimental validation and protein structure analysis, further reveal how HLA-binding motifs change with peptide length and predict new cooperative effects between distant residues in HLA-B07:02 ligands.

Use of HLA peptidomics and whole exome sequencing to identify human immunogenic neo-antigens.

The antigenicity of cells is demarcated by the peptides bound by their Human Leucocyte Antigen (HLA) molecules. Through this antigen presentation, T cell specificity response is controlled. As a fraction of the expressed mutated peptides is presented on the HLA, these neo-epitopes could be immunogenic. Such neo-antigens have recently been identified through screening for predicted mutated peptides, using synthetic peptides or ones expressed from minigenes, combined with screening of patient tumor-infiltrating lymphocytes (TILs). Here we present a time and cost-effective method that combines whole-exome sequencing analysis with HLA peptidome mass spectrometry, to identify neo-antigens in a melanoma patient. Of the 1,019 amino acid changes identified through exome sequencing, two were confirmed by mass spectrometry to be presented by the cells. We then synthesized peptides and evaluated the two mutated neo-antigens for reactivity with autologous bulk TILs, and found that one yielded mutant-specific T-cell response. Our results demonstrate that this method can be used for immune response prediction and promise to provide an alternative approach for identifying immunogenic neo-epitopes in cancer.

Selection of peptide mimics of HIV-1 epitope recognized by neutralizing antibody VRC01.

The ability to induce anti-HIV-1 antibodies that can neutralize a broad spectrum of viral isolates from different subtypes seems to be a key requirement for development of an effective HIV-1 vaccine. The epitopes recognized by the most potent broadly neutralizing antibodies that have been characterized are largely discontinuous. Mimetics of such conformational epitopes could be potentially used as components of a synthetic immunogen that can elicit neutralizing antibodies. Here we used phage display technology to identify peptide motifs that mimic the epitope recognized by monoclonal antibody VRC01, which is able to neutralize up to 91% of circulating primary isolates. Three rounds of biopanning were performed against 2 different phage peptide libraries for this purpose. The binding specificity of selected phage clones to monoclonal antibody VRC01 was estimated using dot blot analysis. The putative peptide mimics exposed on the surface of selected phages were analyzed for conformational and linear homology to the surface of HIV-1 gp120 fragment using computational analysis. Corresponding peptides were synthesized and checked for their ability to interfere with neutralization activity of VRC01 in a competitive inhibition assay. One of the most common peptides selected from 12-mer phage library was found to partially mimic a CD4-binding loop fragment, whereas none of the circular C7C-mer peptides was able to mimic any HIV-1 domains. However, peptides identified from both the 12-mer and C7C-mer peptide libraries showed rescue of HIV-1 infectivity in the competitive inhibition assay. The identification of epitope mimics may lead to novel immunogens capable of inducing broadly reactive neutralizing antibodies.

Antimicrobial peptides and peptidomimetics - potent therapeutic allies for staphylococcal infections.

The pervasiveness of bacterial resistance to conventional antibiotics, particularly those associated with staphylococcal infections, has become a global epidemic. However, research involving antimicrobial peptides (AMPs) and their synthetic analogues has unearthed a potentially novel class of antibacterials for the treatment of an array of diseases caused by pathogenic bacteria, such as staphylococci. AMPs have several unique advantages over traditional antibiotics such as the projected slow emergence of bacterial resistance to these agents and their capability to modulate the host immune response to infection. Unfortunately, their susceptibility to proteolytic degradation, loss of antimicrobial activity due to serum binding or physiological concentration of salts, and toxicity to host tissues has limited their use as systemic agents thus far. Additionally, the presence of economic and regulatory obstacles has hindered the translation of AMPs, as antimicrobials, from the bench to the clinic. The present review delves further into the benefits and challenges of utilizing AMPs as antibacterial agents (particularly for staphylococcal infections), the methods which have been utilized to overcome their limitations, their successes and failures in clinical trials, and future avenues for researchers to pursue to develop AMPs as novel therapeutic allies in the treatment of bacterial infections.

Peptides and peptidomimetics as immunomodulators.

Peptides and peptidomimetics can function as immunomodulating agents by either blocking the immune response or stimulating the immune response to generate tolerance. Knowledge of B- or T-cell epitopes along with conformational constraints is important in the design of peptide-based immunomodulating agents. Work on the conformational aspects of peptides, synthesis and modified amino acid side chains have contributed to the development of a new generation of therapeutic agents for autoimmune diseases and cancer. The design of peptides/peptidomimetics for immunomodulation in autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus and HIV infection is reviewed. In cancer therapy, peptide epitopes are used in such a way that the body is trained to recognize and fight the cancer cells locally as well as systemically.

Dextran as a generally applicable multivalent scaffold for improving immunoglobulin-binding affinities of peptide and peptidomimetic ligands.

Molecules able to bind the antigen-binding sites of antibodies are of interest in medicine and immunology. Since most antibodies are bivalent, higher affinity recognition can be achieved through avidity effects in which a construct containing two or more copies of the ligand engages both arms of the immunoglobulin simultaneously. This can be achieved routinely by immobilizing antibody ligands at high density on solid surfaces, such as ELISA plates, but there is surprisingly little literature on scaffolds that routinely support bivalent binding of antibody ligands in solution, particularly for the important case of human IgG antibodies. Here we show that the simple strategy of linking two antigens with a polyethylene glycol (PEG) spacer long enough to span the two arms of an antibody results in higher affinity binding in some, but not all, cases. However, we found that the creation of multimeric constructs in which several antibody ligands are displayed on a dextran polymer reliably provides much higher affinity binding than is observed with the monomer in all cases tested. Since these dextran conjugates are simple to construct, they provide a general and convenient strategy to transform modest affinity antibody ligands into high affinity probes. An additional advantage is that the antibody ligands occupy only a small number of the reactive sites on the dextran, so that molecular cargo can be attached easily, creating molecules capable of delivering this cargo to cells displaying antigen-specific receptors.

Virosome-formulated Plasmodium falciparum AMA-1 & CSP derived peptides as malaria vaccine: randomized phase 1b trial in semi-immune adults & children.

BACKGROUND: This trial was conducted to evaluate the safety and immunogenicity of two virosome formulated malaria peptidomimetics derived from Plasmodium falciparum AMA-1 and CSP in malaria semi-immune adults and children. METHODS: The design was a prospective randomized, double-blind, controlled, age-deescalating study with two immunizations. 10 adults and 40 children (aged 5-9 years) living in a malaria endemic area were immunized with PEV3B or virosomal influenza vaccine Inflexal®V on day 0 and 90. RESULTS: No serious or severe adverse events (AEs) related to the vaccines were observed. The only local solicited AE reported was pain at injection site, which affected more children in the Inflexal®V group compared to the PEV3B group (p = 0.014). In the PEV3B group, IgG ELISA endpoint titers specific for the AMA-1 and CSP peptide antigens were significantly higher for most time points compared to the Inflexal®V control group. Across all time points after first immunization the average ratio of endpoint titers to baseline values in PEV3B subjects ranged from 4 to 15 in adults and from 4 to 66 in children. As an exploratory outcome, we found that the incidence rate of clinical malaria episodes in children vaccinees was half the rate of the control children between study days 30 and 365 (0.0035 episodes per day at risk for PEV3B vs. 0.0069 for Inflexal®V; RR  = 0.50 [95%-CI: 0.29-0.88], p = 0.02). CONCLUSION: These findings provide a strong basis for the further development of multivalent virosomal malaria peptide vaccines. TRIAL REGISTRATION: ClinicalTrials.gov NCT00513669.

Generation of a unique small molecule peptidomimetic that neutralizes lupus autoantibody activity.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of pathogenic autoantibodies, many of which are directed against nuclear antigens, in particular double-stranded (ds) DNA. Both clinical studies and animal models have shown that anti-dsDNA antibodies contribute to kidney disease, which is present in 50% of lupus patients and is a major cause of mortality. We previously demonstrated that a subset of nephrotoxic anti-dsDNA antibodies also recognizes the pentapeptide consensus sequence D/E W D/E Y S/G (DWEYS) present in the NR2A and NR2B subunits of the N-methyl-d-aspartate receptor (NMDAR). Autoantibodies with this specificity are present in ≈40% of lupus patient sera and are both nephrotoxic and neurotoxic. Elevated titers are present in cerebrospinal fluid of patients with central nervous system manifestations of SLE. Administration of the nonnaturally occurring D form of the DWEYS pentapeptide prevents these antibodies from depositing in glomeruli and from mediating neuronal excitotoxicity. To craft a more useful therapeutic, we used the structural features of the DWEYS peptide to design a unique, selective, and potent small molecule peptidomimetic, FISLE-412, which neutralizes anti-dsDNA/NMDAR lupus autoantibodies and prevents their pathogenic interaction with tissue antigens. This compound, or others derived from it, may provide a unique strategy for the development of lupus therapeutics.

Vaccination with viral protein-mimicking peptides postpones mortality in domestic pigs infected by African swine fever virus.

Periodic outbreaks of African swine fever virus (ASFV) infection around the world threaten local populations of domestic pigs with lethal disease and provide grounds for pandemic spread. Effective vaccination may bring this threat under control. We investigated the effectiveness of select peptides mimicking viral proteins in establishing a protective immune response. Forty-six synthetic peptides based on the analysis of the complete nucleotide sequence of ASFV were tested for immunogenicity in mice. The 17 best immune response-inducing peptide candidates were selected for further investigation. Twenty-four domestic pigs, 3-4 months old and weighing 20-25 kg, were divided into six groups (n = 4) and immunized by subcutaneous injection using a standard three-round injection protocol with one of four peptide combinations prepared from the 17 peptides (Groups 1-4) or with carrier only (Group 5). Group 6, the control, was not vaccinated. Animal body temperature and behavior were monitored during and post immunization for health assessment. Two weeks after the last round of immunizations, the pigs were infected with live ASFV (Espania 70) at 6.0 Ig GAE50/cm3, and the survival rate was monitored. Blood samples were collected for analysis the day before infection and on days 3, 7 and 10 post-infection, or from deceased animals. The serum titers of specific immunoglobulins against synthetic peptides and whole inactivated ASFV were determined by enzyme immunoassay before and after infection. The presence of viral DNA in blood serum samples was determined by polymerase chain reaction. Viral infection activity in blood sera was determined by heme absorption in cultured porcine bone marrow and porcine leukocyte cells. Repeating the injection of synthetic peptides in both the mice and pigs produced an immune response specific to individual peptides, which differed widely in the intensity scale. Specific anti-whole virus immunoglobulin binding activity in the swine serum samples from all groups was below the detection limit. Viral DNA was positively identified in all the samples infected with viral preparations. Viral infection activity was present in all the infected animals and steadily increased with time. On day 3 after infection, the viral titer was significantly lower in Groups 1 and 3 than in the unvaccinated controls. In deceased animals, the viral titer was significantly lower in Groups 1 and 3 than in the controls. All infected animals died within 17 days of infection. The average survival rate was significantly higher in Groups 1 and 3 (12.0 and 14.3 days, respectively) than in the controls (9.8 days). Vaccination with specific synthetic peptides significantly delayed mortality in domestic pigs infected with ASFV. These results justify further investigation aimed at developing an effective vaccine against ASFV infection.

Peptidomimetics: modifying peptides in the pursuit of better vaccines.

Peptide vaccines represent a potential strategy for the prevention and treatment of pathogenic diseases, cancers and autoimmune disorders; their low cost, ease of synthesis and inherent safety are all attractive features. However, they have remained largely unsuccessful owing to low immunogenicity, predominantly stemming from reduced bioavailability due to rapid proteolytic degradation in vivo. The field of peptidomimetics, through chemical alteration of epitope structure, attempts to modify peptides to engender improved immunogenicity and stability, with the ultimate aim of rationally designing more efficacious vaccines. This article discusses the strategies employed in this process and recent advances within the field.

Study of various presentation forms for a peptide mimetic of Neisseria meningitidis serogroup B capsular polysaccharide.

The formulation of a broadly protective vaccine to prevent the serogroup B Neisseria meningitidis (MenB) disease is still an unmet medical need. We have previously reported the induction of bactericidal and protective antibodies against MenB after immunization of mice with a phage-displayed peptide named 4 L-5. This peptide mimics a capsular polysaccharide (CPS) epitope in MenB. With the aim of developing vaccine formulations that could be used in humans, we evaluate in this study various forms of presentation to the immune system of the 4 L-5 sequence, based on synthetic peptides. We synthesized the following: (i) a linear 4 L-5 peptide, (ii) a multiple antigen peptide containing four copies of the 4 L-5 sequence (named MAP), which was then dimerized, and the product named dimeric MAP, and (iii) a second multiple antigen peptide, in this case with two copies of the 4 L-5 sequence and a copy of a T-helper cell epitope of tetanus toxoid, which was then dimerized and the product named MAP-TT. The linear peptide, the MAP, and the dimeric MAP were conjugated to the carrier protein P64K by different conjugation methods. Plain antigens and antigens coupled to P64K were used to immunize BALB/c mice. Of those variants that gave immunogenic results, MAP-TT rendered the highest levels of specific antipeptide IgG antibodies and serum bactericidal activity. These results can find application in the development of meningococcal vaccine candidates and in peptide-based vaccines strategies.

Development of designed site-directed pseudopeptide-peptido-mimetic immunogens as novel minimal subunit-vaccine candidates for malaria.

Synthetic vaccines constitute the most promising tools for controlling and preventing infectious diseases. When synthetic immunogens are designed from the pathogen native sequences, these are normally poorly immunogenic and do not induce protection, as demonstrated in our research. After attempting many synthetic strategies for improving the immunogenicity properties of these sequences, the approach consisting of identifying high binding motifs present in those, and then performing specific changes on amino-acids belonging to such motifs, has proven to be a workable strategy. In addition, other strategies consisting of chemically introducing non-natural constraints to the backbone topology of the molecule and modifying the α-carbon asymmetry are becoming valuable tools to be considered in this pursuit. Non-natural structural constraints to the peptide backbone can be achieved by introducing peptide bond isosters such as reduced amides, partially retro or retro-inverso modifications or even including urea motifs. The second can be obtained by strategically replacing L-amino-acids with their enantiomeric forms for obtaining both structurally site-directed designed immunogens as potential vaccine candidates and their Ig structural molecular images, both having immuno-therapeutic effects for preventing and controlling malaria.