Design Of Synthetic Vaccines Research Articles

Fully synthetic Tn-based three-component cancer vaccine using covalently linked TLR4 ligand MPLA and iNKT cell agonist KRN-7000 as built-in adjuvant effectively protects mice from tumor development.

We present a new strategy for self-adjuvanting vaccine development that has different types of covalently-linked immunostimulants as the carrier molecule. Using Tn antigen as the model, a three-component vaccine (MPLA-Tn-KRN7000) containing the TLR4 ligand MPLA and the iNKT cell agonist KRN7000 was designed and synthesized. This expands fully synthetic self-adjuvanting vaccine studies that use a single carrier to one with two different types of carriers. The corresponding two-component conjugate vaccines Tn-MPLA, Tn-KRN7000 and Tn-CRM197 were also synthesized, as controls. The immunological evaluation found that MPLA-Tn-KRN7000 elicits robust Tn-specific and T cell-dependent immunity. The antibodies specifically recognized, bound to and exhibited complement-dependent cytotoxicity against Tn-positive cancer cells. In addition, MPLA-Tn-KRN7000 increased the survival rate and survival time of tumor-challenged mice, and surviving mice reject further tumor attacks without any additional treatment. Compared to the glycoprotein vaccine Tn-CRM197, the two-component conjugate vaccines, Tn-MPLA and Tn-KRN7000, and the physical mixture of Tn-MPLA and Tn-KRN7000, MPLA-Tn-KRN7000 showed the most effect at combating tumor cells both invitro and invivo. The comparison of immunological studies in wild-type and TLR4 knockout mice, along with the test of binding affinity to CD1d protein suggests that the covalently linked MPLA-KRN7000 immunostimulant induces a synergistic activation of TLR4 and iNKT cell that improves the immunogenicity of Tn. This work demonstrates that MPLA-Tn-KRN7000 has the potential to be a vaccine candidate and provides a new direction for fully synthetic vaccine design.

Bioinformatics pipeline unveils genetic variability to synthetic vaccine design for Indian SARS-CoV-2 genomes

In the worrisome scenarios of various waves of SARS-CoV-2 pandemic, a comprehensive bioinformatics pipeline is essential to analyse the virus genomes in order to understand its evolution, thereby identifying mutations as signature SNPs, conserved regions and subsequently to design epitope based synthetic vaccine. We have thus performed multiple sequence alignment of 4996 Indian SARS-CoV-2 genomes as a case study using MAFFT followed by phylogenetic analysis using Nextstrain to identify virus clades. Furthermore, based on the entropy of each genomic coordinate of the aligned sequences, conserved regions are identified. After refinement of the conserved regions, based on its length, one conserved region is identified for which the primers and probes are reported for virus detection. The refined conserved regions are also used to identify T-cell and B-cell epitopes along with their immunogenic and antigenic scores. Such scores are used for selecting the most immunogenic and antigenic epitopes. By executing this pipeline, 40 unique signature SNPs are identified resulting in 23 non-synonymous signature SNPs which provide 28 amino acid changes in protein. On the other hand, 12 conserved regions are selected based on refinement criteria out of which one is selected as the potential target for virus detection. Additionally, 22 MHC-I and 21 MHC-II restricted T-cell epitopes with 10 unique HLA alleles each and 17 B-cell epitopes are obtained for 12 conserved regions. All the results are validated both quantitatively and qualitatively which show that from genetic variability to synthetic vaccine design, the proposed pipeline can be used effectively to combat SARS-CoV-2.

Techniques assisting peptide vaccine and peptidomimetic design. Sidechain exposure in the SARS-CoV-2 spike glycoprotein

The aim of the present study is to discuss the design of peptide vaccines and peptidomimetics against SARS-COV-2, to develop and apply a method of protein structure analysis that is particularly appropriate to applying and discussing such design, and also to use that method to summarize some important features of the SARS-COV-2 spike protein sequence. A tool for assessing sidechain exposure in the SARS-CoV-2 spike glycoprotein is described. It extends to assessing accessibility of sidechains by considering several different three-dimensional structure determinations of SARS-CoV-2 and SARS-CoV-1 spike protein. The method is designed to be insensitive to a distance limit for counting neighboring atoms and the results are in good agreement with the physical chemical properties and exposure trends of the 20 naturally occurring sidechains. The spike protein sequence is analyzed with comment regarding exposable character. It includes studies of complexes with antibody elements and ACE2. These indicate changes in exposure at sites remote to those at which the antibody binds. They are of interest concerning design of synthetic peptide vaccines, and for peptidomimetics as a basis of drug discovery. The method was also developed in order to provide linear (one-dimensional) information that can be used along with other bioinformatics data of this kind in data mining and machine learning, potentially as genomic data regarding protein polymorphisms to be combined with more traditional clinical data.

Prevalence of antibodies against a cyclic peptide mimicking the FG loop of the human papillomavirus type 16 capsid among Tunisian women

BackgroundIn the past decade, cervical cancer has gone from being the second to the fourth most common cancer in women worldwide, but remains the second most common in developing countries. This cancer is most commonly caused by high-risk types of human papillomavirus (HPV), mainly type 16 (HPV16), which are sexually transmitted. This study aimed to investigate the usefulness of a cyclic synthetic peptide designed from the major L1 capsid protein of HPV16 for detecting anti-HPV16 antibodies.MethodsWe designed and synthetized a peptide that corresponds to the full sequence of the surface-exposed FG loop. We tested the antigenicity of the linear and the cyclic peptides against HPV16 L1 monoclonal antibodies. We used ELISA to detect anti-peptide antibodies in sera and cervical secretions of 179 Tunisian women, and we applied polymerase chain reaction and direct sequencing methods to detect and genotype HPV DNA.ResultsBoth the linear and the cyclic peptides were recognized by the same neutralizing monoclonal antibodies, but the cyclic peptide was more reactive with human sera. The prevalence of the anti-peptide antibodies in sera was higher in women with low-grade squamous intraepithelial lesions (LGSIL) than in women with high-grade squamous intraepithelial lesions (HGSIL) (44% and 15%, respectively). This contrasts with HPV16 DNA prevalence. Compared to women from the general population, systemic IgG prevalence was significantly higher among sex workers (25%; P = 0.002) and women with LGSIL (44%; P = 0.001). In addition, systemic IgA and cervical IgG prevalence was higher among sex workers only (P = 0.002 and P = 0.001, respectively). We did not observe anti-peptide IgG antibodies in women with a current HPV16 infection.ConclusionAnti-peptide IgG in sera or in cervical secretions could be markers of an effective natural immunization against HPV16. This may open novel perspectives for monitoring vaccinated women and for the design of synthetic peptide-based vaccines.

PVACtools: A Computational Toolkit to Identify and Visualize Cancer Neoantigens.

Identification of neoantigens is a critical step in predicting response to checkpoint blockade therapy and design of personalized cancer vaccines. This is a cross-disciplinary challenge, involving genomics, proteomics, immunology, and computational approaches. We have built a computational framework called pVACtools that, when paired with a well-established genomics pipeline, produces an end-to-end solution for neoantigen characterization. pVACtools supports identification of altered peptides from different mechanisms, including point mutations, in-frame and frameshift insertions and deletions, and gene fusions. Prediction of peptide:MHC binding is accomplished by supporting an ensemble of MHC Class I and II binding algorithms within a framework designed to facilitate the incorporation of additional algorithms. Prioritization of predicted peptides occurs by integrating diverse data, including mutant allele expression, peptide binding affinities, and determination whether a mutation is clonal or subclonal. Interactive visualization via a Web interface allows clinical users to efficiently generate, review, and interpret results, selecting candidate peptides for individual patient vaccine designs. Additional modules support design choices needed for competing vaccine delivery approaches. One such module optimizes peptide ordering to minimize junctional epitopes in DNA vector vaccines. Downstream analysis commands for synthetic long peptide vaccines are available to assess candidates for factors that influence peptide synthesis. All of the aforementioned steps are executed via a modular workflow consisting of tools for neoantigen prediction from somatic alterations (pVACseq and pVACfuse), prioritization, and selection using a graphical Web-based interface (pVACviz), and design of DNA vector-based vaccines (pVACvector) and synthetic long peptide vaccines. pVACtools is available at http://www.pvactools.org.

Enabling sublingual peptide immunization with molecular self-assemblies

Short peptides are poorly immunogenic when delivered sublingually – under the tongue. Nanomaterial delivery of peptides could be utilized to improve immunogenicity towards designed sublingual vaccines, but nanomaterials have not been widely successful in sublingual vaccines owing to the challenges of transport through the sublingual mucosa. Here, we report that the sublingual immunogenicity of peptides is negligible, even in the presence of sublingual adjuvants or when PEGylated, but can be dramatically enhanced by assembly into supramolecular polymer-peptide nanofibers bearing low-molecular weight PEG, optimally between 2000 and 3000 Da. Neither PEGylation nor a sublingual adjuvant were capable of rendering peptides immunogenic without assembly into nanofibers. We found that PEG decreased nanofiber interactions with mucin and promoted longer residence time at the sublingual immunization site. Parallel investigations with shortened nanofibers indicated that the size of the assemblies had a surprisingly negligible influence over sublingual immunogenicity. In mice, optimized formulations were capable of raising strong and highly durable systemic antibody responses, antibodies in the upper respiratory and reproductive tracts, and systemic antigen-specific T-cell responses. These nanofiber-based sublingual vaccines were effective with both protein and nucleotide adjuvants and raised responses against both a model peptide epitope and a peptide epitope from M. tuberculosis. Further, PASylation (modification of nanofibers with peptide sequences rich in Pro, Ala, and Ser) could be substituted for PEGylation to also achieve sublingual immunogenicity. These findings indicated that surface properties supersede nanomaterial size in modulating sublingual nanomaterial immunogenicity, having important implications for the design of synthetic sublingual vaccines.

Extensive genomic recoding by codon-pair deoptimization selective for mammals is a flexible tool to generate attenuated vaccine candidates for dengue virus 2

The four serotypes of dengue virus (DENV) are the leading etiologic agent of disease caused by arthropod-borne viruses (arboviruses) in the world, with billions at risk of DENV infection spread by infected mosquitoes. DENV causes illness ranging from dengue fever (DF) to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DENV proliferates well in two different host systems, an invertebrate mosquito vector and vertebrate primate host, which have a distinct difference in their preference of codon pairs (CP) for translation (different “codon pair bias”). Consequently, arboviruses must delicately balance the use of codon pairs between mammals and arthropods, which presents an Achilles’ heel that we have exploited by specifically shifting the codon pair preference in the E and NS3 ORFs away from mammals while keeping the CPB favorable for mosquito ORFs. Here we report that recoding of the ORFs has led to variants that were over-attenuated in rhesus macaques although induction of protective antibodies in animals vaccinated with the smallest recoded ORF (E) was observed. The flexibility of our synthetic vaccine design (by decreasing the number of unfavorable CPs in the E ORF), allowed us to construct two new vaccine candidates (EhminA and EhminB) with intermediate attenuation in cell culture and neonatal mice, a result demonstrating proof of concept. New DENV vaccine candidates are being developed based on selective attenuation by dramatic recoding, with flexibility in balancing the attenuation and immunogenicity by marrying rational design and empirical modification.

Antigen structure affects cellular routing through DC-SIGN

Dendritic cell (DC) lectins mediate the recognition, uptake, and processing of antigens, but they can also be coopted by pathogens for infection. These distinct activities depend upon the routing of antigens within the cell. Antigens directed to endosomal compartments are degraded, and the peptides are presented on major histocompatibility complex class II molecules, thereby promoting immunity. Alternatively, HIV-1 can avoid degradation, as virus engagement with C-type lectin receptors (CLRs), such as DC-SIGN (DC-specific ICAM-3-grabbing nonintegrin) results in trafficking to surface-accessible invaginated pockets. This process appears to enable infection of T cells in trans We sought to explore whether antigen fate upon CLR-mediated internalization was affected by antigen physical properties. To this end, we employed the ring-opening metathesis polymerization to generate glycopolymers that each display multiple copies of mannoside ligand for DC-SIGN, yet differ in length and size. The rate and extent of glycopolymer internalization depended upon polymer structure-longer polymers were internalized more rapidly and more efficiently than were shorter polymers. The trafficking, however, did not differ, and both short and longer polymers colocalized with transferrin-labeled early endosomes. To explore how DC-SIGN directs larger particles, such as pathogens, we induced aggregation of the polymers to access particulate antigens. Strikingly, these particulate antigens were diverted to the invaginated pockets that harbor HIV-1. Thus, antigen structure has a dramatic effect on DC-SIGN-mediated uptake and trafficking. These findings have consequences for the design of synthetic vaccines. Additionally, the results suggest strategies for targeting DC reservoirs that harbor viral pathogens.

Polyester as Antigen Carrier toward Particulate Vaccines.

Spherical polyhydroxyalkanoate (PHA) inclusions are naturally self-assembled inside bacteria. These PHA beads are shell-core structures composed of a hydrophobic PHA core surrounded by proteins, such as PHA synthase (PhaC). PhaC is covalently attached and serves as an anchor protein for foreign protein vaccine candidate antigens. PHA beads displaying single and multiple antigens showed enhanced immunological properties when compared to respective soluble vaccines. This review highlights the unique design space of the PHA bead-based vaccines toward the development of safe and synthetic particulate vaccines. The PHA bead technology will be compared with chemically synthesized polyesters, such as polylactic acids, formulated to deliver vaccine antigens. The performance of PHA bead vaccine candidates to induce specific immune responses and protective immunity against bacterial and viral pathogens in animal trials will be summarized. We propose that the PHA bead technology offers a versatile vaccine platform for design and cost-effective manufacture of synthetic multivalent vaccines.

Characterization and Design of Synthetic Vaccines

Characterization and Design of Synthetic Vaccines

Epidemics and Peptide Vaccine Response: A Brief Review.

We briefly review the situations arising out of epidemics that erupt rather suddenly, threatening life and livelihoods of humans. Ebola, Zika and the Nipah virus outbreaks are recent examples where the viral epidemics have led to considerably high degree of fatalities or debilitating consequences. The problems are accentuated by a lack of drugs or vaccines effective against the new and emergent viruses, and the inordinate amount of temporal and financial resources that are required to combat the novel pathogens. Progress in computational, biological and informational sciences have made it possible to consider design of synthetic vaccines that can be rapidly developed and deployed to help stem the damages. In this review, we consider the pros and cons of this new paradigm and suggest a new system where the manufacturing process can be decentralized to provide more targeted vaccines to meet the urgent needs of protection in case of a rampaging epidemic.

Correction: Development of a novel immunoproteasome digestion assay for synthetic long peptide vaccine design.

[This corrects the article DOI: 10.1371/journal.pone.0199249.].

Development of a novel immunoproteasome digestion assay for synthetic long peptide vaccine design.

Recently, many autologous tumor antigens have been examined for their potential use in cancer immunotherapy. However, the success of cancer vaccines in clinical trials has been limited, partly because of the limitations of using single, short peptides in most attempts. With this in mind, we aimed to develop multivalent synthetic long peptide (SLP) vaccines containing multiple cytotoxic T-lymphocyte (CTL) epitopes. However, to confirm whether a multivalent vaccine can induce an individual epitope-specific CTL, the only viable screening strategies currently available are interferon-gamma (IFN-μ enzyme-linked immunospot (ELISPOT) assays using human peripheral blood mononuclear cells, or expensive human leukocyte antigen (HLA)-expressing mice. In this report, we evaluated the use of our developed murine-20S immunoproteasome (i20S) digestion assay, and found that it could predict the results of IFN-μ ELISPOT assays. Importantly, the murine-i20S digestion assay not only predicted CTL induction, but also antitumor activity in an HLA-expressing mouse model. We conclude that the murine-i20S digestion assay is an extremely useful tool for the development of “all functional” multivalent SLP vaccines.

Synthetic Vaccine Characterization and Design

Synthetic Vaccine Characterization and Design

NKT-cell glycolipid agonist as adjuvant in synthetic vaccine

NKT cells are CD1d-restricted, glycolipid antigen-reactive, immunoregulatory T lymphocytes that can serve as a bridge between the innate and adaptive immunities. NKT cells have a wide range of therapeutic application in autoimmunity, transplant biology, infectious disease, cancer, and vaccinology. Rather than triggering “danger signal” and eliciting an innate immune response, αGalCer-based NKT-cell agonist act via a unique mechanism, recruiting NKT cells which play a T helper-like role even without peptide as Th epitope. Importantly, the non-polymorphism of CD1d render glycolipid a universal helper epitope, offering the potential to simplify the vaccine construct capable of eliciting consistent immune response in different individuals. This review details recent advances in the design of synthetic vaccines using NKT-cell agonist as adjuvant, highlighting the role of organic synthesis and conjugation technique to enhance the immunological actives and to simplify the vaccine constructs.

An Approach for a Synthetic CTL Vaccine Design against Zika Flavivirus Using Class I and Class II Epitopes Identified by Computer Modeling.

The threat posed by severe congenital abnormalities related to Zika virus (ZKV) infection during pregnancy has turned development of a ZKV vaccine into an emergency. Recent work suggests that the cytotoxic T lymphocyte (CTL) response to infection is an important defense mechanism in response to ZKV. Here, we develop the rationale and strategy for a new approach to developing cytotoxic T lymphocyte (CTL) vaccines for ZKV flavivirus infection. The proposed approach is based on recent studies using a protein structure computer model for HIV epitope selection designed to select epitopes for CTL attack optimized for viruses that exhibit antigenic drift. Because naturally processed and presented human ZKV T cell epitopes have not yet been described, we identified predicted class I peptide sequences on ZKV matching previously identified DNV (Dengue) class I epitopes and by using a Major Histocompatibility Complex (MHC) binding prediction tool. A subset of those met the criteria for optimal CD8+ attack based on physical chemistry parameters determined by analysis of the ZKV protein structure encoded in open source Protein Data File (PDB) format files. We also identified candidate ZKV epitopes predicted to bind promiscuously to multiple HLA class II molecules that could provide help to the CTL responses. This work suggests that a CTL vaccine for ZKV may be possible even if ZKV exhibits significant antigenic drift. We have previously described a microsphere-based CTL vaccine platform capable of eliciting an immune response for class I epitopes in mice and are currently working toward in vivo testing of class I and class II epitope delivery directed against ZKV epitopes using the same microsphere-based vaccine.

Protein Epitope Mimetics: From New Antibiotics to Supramolecular Synthetic Vaccines.

Protein epitope mimetics provide powerful tools to study biomolecular recognition in many areas of chemical biology. They may also provide access to new biologically active molecules and potentially to new classes of drug and vaccine candidates. Here we highlight approaches for the design of folded, structurally defined epitope mimetics, by incorporating backbone and side chains of hot residues onto a stable constrained scaffold. Using robust synthetic methods, the structural, biological, and physical properties of epitope mimetics can be optimized, by variation of both side chain and backbone chemistry. To illustrate the potential of protein epitope mimetics in medicinal chemistry and biotechnology, we present studies in two areas of infectology; the discovery of new antibiotics targeting essential outer membrane (OM) proteins in Gram-negative bacteria and the design of supramolecular synthetic vaccines. The discovery of new antibiotics with novel mechanisms of action, in particular to combat infections caused by Gram-negative pathogens, represents a major challenge in medicinal chemistry. We were inspired by naturally occurring cationic antimicrobial peptides to design structurally related peptidomimetics and to optimize their antimicrobial properties through library synthesis and screening. Through these efforts, we could show that antimicrobial β-hairpin mimetics may have structures and properties that facilitate interactions with essential bacterial β-barrel OM proteins. One recently discovered family of antimicrobial peptidomimetics targets the β-barrel protein LptD in Pseudomonas spp. This protein plays a key role in lipopolysaccaride (LPS) transport to the cell surface during OM biogenesis. Through a highly selective interaction with LptD, the peptidomimetic blocks LPS transport, resulting in nanomolar antimicrobial activity against the important human pathogen P. aeruginosa. Epitope mimetics may also have great potential in the field of vaccinology, where structural information on complexes between neutralizing antibodies and their cognate epitopes can be taken as a starting point for B cell epitope mimetic design. In order to generate potent immune responses, an effective method of delivering epitope mimetics to relevant cells and tissues in the immune system is also required. For this, engineered synthetic nanoparticles (synthetic virus-like particles, SVLPs) prepared using supramolecular chemistry can be designed with optimal surface properties for efficient dendritic cell-mediated delivery of folded B-cell and linear T-cell epitopes, along with ligands for pattern recognition receptors, into lymphoid tissues. In this way, multivalent display of the epitope mimetics occurs over the surface of the nanoparticle, suitable for cross-linking B cell receptors. In this highly immunogenic format, strong epitope-specific humoral immune responses can be elicited that target infections caused by pathogenic microorganisms. Other potential applications of epitope mimetics in next-generation therapeutics are also discussed.

Deciphering Antigenic Determinants of Streptococcus pneumoniae Serotype 4 Capsular Polysaccharide using Synthetic Oligosaccharides.

Streptococcus pneumoniae is a major cause of mortality and morbidity worldwide. More than 90 S. pneumoniae serotypes are distinguished based on the structure of their primary targets to the human immune system, the capsular polysaccharides (CPSs). The CPS of the prevalent serotype 4 (ST4) is composed of tetrasaccharide repeating units and is included in existing pneumococcal vaccines. Still, the structural antigenic determinants that are essential for protective immunity, including the role of the rare and labile cyclic trans-(2,3) pyruvate ketal modification, remain largely unknown. Molecular insights will support the design of synthetic subunit oligosaccharide vaccines. Here, we identified the key antigenic determinants of ST4 CPS with the help of pyruvated and nonpyruvated synthetic repeating unit glycans. Glycan arrays revealed oligosaccharide antigens recognized by antibodies in the human reference serum. Selected depyruvated ST4 oligosaccharides were used to formulate neoglycoconjugates and immunologically evaluated in mice. These oligosaccharides were highly immunogenic, but the resulting antiglycan antibodies showed only limited binding to the natural CPS present on the bacterial surface. Glycan array and surface plasmon resonance analysis of murine polyclonal serum antibodies as well as monoclonal antibodies revealed that terminal sugars are important in directing the immune responses. The pyruvate modification on the oligosaccharide is needed for cross-reactivity with the native CPS. These findings are an important step toward the design of oligosaccharide-based vaccines against S. pneumoniae ST4.

Assembly and Assessment of DNA Scaffolded Vaccines.

Vaccines play an important role in preventing many life-threatening infectious diseases. To meet the demand of vaccination for treating a wide range of diseases, rational vaccine design has been recognized as a desirable and necessary strategy for development of safe and effective vaccines. DNA nanostructures are advantageous in the design and construction of synthetic vaccines, owing to their robust self-assembly, programmability, and precision control in complex organization, as well as their intrinsic adjuvant activity. Here, we describe a modular assembly of DNA scaffolded vaccine complex, composing of a model antigen, streptavidin, and adjuvant, CpG oligonucleotide. The DNA-assembled vaccines were found to elicit strong antigen-specific antibody responses, but causing little or no adverse reactions. Conceivably, this vaccine platform can be further optimized for improved immunogenicity and extended to the construction of various subunit vaccines.

Recent advances in the molecular design of synthetic vaccines.

Vaccines have typically been prepared using whole organisms. These are normally either attenuated bacteria or viruses that are live but have been altered to reduce their virulence, or pathogens that have been inactivated and effectively killed through exposure to heat or formaldehyde. However, using whole organisms to elicit an immune response introduces the potential for infections arising from a reversion to a virulent form in live pathogens, unproductive reactions to vaccine components or batch-to-batch variability. Synthetic vaccines, in which a molecular antigen is conjugated to a carrier protein, offer the opportunity to circumvent these problems. This Perspective will highlight the progress that has been achieved in developing synthetic vaccines using a variety of molecular antigens. In particular, the different approaches used to develop conjugate vaccines using peptide/proteins, carbohydrates and other small molecule haptens as antigens are compared.