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The current pandemic called COVID-19 caused by the SARS-CoV-2 virus brought the need for the search for fast alternatives to both control and fight the SARS-CoV-2 infection. Therefore, a race for a vaccine against COVID-19 took place, and some vaccines have been approved for emergency use in several countries in a record time. Ongoing prophylactic research has sought faster, safer, and precise alternatives by redirecting knowledge of other vaccines, and/or the development of new strategies using available tools, mainly in the areas of genomics and bioinformatics. The current review highlights the development of synthetic antigen vaccines, focusing on the usage of bioinformatics tools for the selection and construction of antigens on the different vaccine constructions under development, as well as strategies to optimize vaccines for COVID-19.

Advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed by non-enzymatic reaction between reducing-sugar and Arg/Lys in proteins and are involved in various diabetic complications. GA-pyridine is derived from glycolaldehyde and is one of the most cytotoxic AGEs. Here, we established a single-chain Fv (scFv) antibody against GA-pyridine, 73MuL9-scFv, and examined the details of its specificity and antigen recognition by using various techniques involving biophysics, chemical biology and structural biology. We also synthesized several compounds that differ slightly in regard to the position and number of GA-pyridine substituent groups, and revealed that GA-pyridine was specifically bound to 73MuL9-scFv. Thermodynamic analysis revealed that the association of GA-pyridine to 73MuL9-scFv was an exothermic and enthalpy driven reaction, and thus that the antigen recognition involved multiple specific interactions. Crystallographic analysis of the Fv fragment of 73MuL9-scFv revealed that several CH-π and hydrogen bond interactions took place between the Fv-fragment and GA-pyridine, which was consistent with the results of thermodynamic analysis. Further studies using 73MuL9-scFv as a tool to clarify the relevance of GA-pyridine to diabetic complications are warranted.

Background Plasmodium vivax proteins with variant interspersed repeats (VIR) are the key proteins used by the parasite to escape from the host immune system through the creation of antigenic variations. However, few studies have been done to elucidate their role as targets of immunity. Thus, this study evaluated the naturally-acquired immune response against VIR proteins in vivax malaria-infected individuals in the Republic of Korea (ROK). Methods Seven recombinant VIR proteins and two synthetic peptides previously studied in other countries that elicited a robust immune response were used to investigate the antibody and cellular immune response in 681 P. vivax-infected people in ROK. The expression of IgM, IgG, and IgG subclasses against each VIR antigen or against PvMSP1-19 was analysed by ELISA. PvMSP1-19, known as a promising vaccine candidate of P. vivax, was used as the positive control for immune response assessment. Furthermore, the cellular immune response to VIR antigens was evaluated by in vitro proliferative assay, cellular activation assay, and cytokine detection in mononuclear cells of the P. vivax-infected population. Results IgM or IgG were detected in 52.4% of the population. Among all the VIR antigens, VIR25 elicited the highest humoral immune response in the whole population with IgG and IgM prevalence of 27.8% and 29.2%, respectively, while PvMSP1-19 elicited even higher prevalence (92%) of IgG in the population. As for the cellular immune response, VIR-C2, PvLP2, and PvMSP1-19 induced high cell activation and secretion of IL-2, IL-6, IL-10, and G-CSF in mononuclear cells from the P. vivax-infected population, comparable with results from PvMSP1-19. However, no significant proliferation response to these antigens was observed between the malaria-infected and healthy groups. Conclusion Moderate natural acquisition of antibody and cellular responses in P. vivax-infected Korean malaria patients presented here are similar to that in other countries. It is interesting that the immune response to VIR antigens is conserved among malaria parasites in different countries, considering that VIR genes are highly polymorphic. This thus warrants further studies to elucidate molecular mechanisms by which human elicit immune response to the malaria parasite VIR antigens.

Cancer immunotherapy has received increasing attention since the success of CTLA-4 and programmed death-1 (PD-1) immune checkpoint inhibitors and CAR-T cells. One of the most promising next-generation cancer treatments is adoptive transfer of immune effector cells. Developing an efficacious adoptive transfer therapy requires growing large numbers of highly purified immune effector cells in a short period of time. γδ T cells can be effectively expanded using synthetic antigens such as pyrophosphomonoesters and nitrogen-containing bisphosphonates (N-BPs). Pyrophosphomonoester antigens, initially identified in mycobacterial extracts, were used for this purpose in the early years of the development of γδ T cell-based therapy. GMP-grade N-BPs, which are now commercially available, are used in many clinical trials worldwide. In order to develop N-BPs for cancer immunotherapy, N-BP prodrugs have been synthesized; among these, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate (PTA) is the most potent compound for stimulating γδ T cells. The activated γδ T cells express high levels of PD-1, suggesting the potential for a combination therapy harnessing γδ T cells and PD-1 immune checkpoint inhibitors. In addition, the functions of γδ T cells can be modified by IL-18. Collectively, the recent findings show that γδ T cells are one of the most promising immune effector subsets for the development of novel cancer immunotherapy.

A vaccine is an immunogen, the administration of which is intended to stimulate the immune system to result in the prevention, amelioration, or therapy of any disease or infection (US Food and Drug Administration. Guidance for Industry: content and format of chemistry, manufacturing, and controls information and establishment description information for a vaccine or related product). A vaccine may be a live attenuated preparation of microorganisms, inactivated (killed) whole organisms, living irradiated cells, crude fractions, or purified immunogens, including those derived from recombinant DNA in a host cell, conjugates formed by covalent linkage of components, synthetic antigens, polynucleotides (such as the plasmid DNA vaccines), living vectored cells expressing specific heterologous immunogens, or cells pulsed with immunogen. Vaccines are highly complex products that differ from small molecule drugs because of the biological nature of the source materials such as those derived from microorganisms as well as the various cell substrates from which some are derived. Regardless of the technology used, because of their complexities, vaccines must undergo extensive characterization and testing. Special expertise and procedures are needed for their manufacture, control, and regulation. The Food and Drug Administration (FDA) is the National Regulatory Authority (NRA) in the United States responsible for assuring quality, safety, and effectiveness of all human medical products, including vaccines for human use.The Center for Biologics Evaluation and Research (CBER) within the US FDA is responsible for overseeing the regulation of therapeutic and preventative vaccines against infectious diseases. Authority for the regulation of vaccines resides in Section 351 of the Public Health Service Act and specific sections of the Federal Food, Drug, and Cosmetic Act (FD&C). Vaccines are regulated as biologics and licensed based on the demonstration of safety and effectiveness. The vaccine development process can be divided into two major categories: those events that are not under the regulatory authority of the FDA and are exploratory in nature and those events that are subject to regulatory authority by the FDA. Exploratory events or research and development cover basic research drug discovery processes that occur before the sponsor submits an investigational new drug application (IND) to the FDA. There are four main stages of vaccine development under the purview of regulatory authorities: preclinical, clinical (IND), licensing, and post-licensure. Throughout their life cycle from preclinical evaluation to post-licensure lot release testing, vaccines are subject to rigorous testing and oversight by manufacturers and NRAs. In this chapter an overview of the regulatory evaluation and testing requirements for vaccines is presented.

Synthetic biology enables the engineering of interactions between living medicines to overcome the specific limitations of any singular therapy. One major challenge of tumor-antigen targeting therapies like chimeric antigen receptor (CAR)-T cells is the identification of targetable antigens that are specifically and uniformly expressed on heterogenous solid tumors. In contrast, certain species of bacteria selectively colonize immune-privileged tumor cores and can be readily engineered as antigen-independent platforms for therapeutic delivery. Bridging these approaches, we develop a platform of probiotic-guided CAR-T cells (ProCARs), in which T cells are engineered to sense synthetic antigens (SA) that are produced and released by tumor-colonizing probiotic bacteria. We demonstrate increased CAR-T cell activation and tumor-cell lysis when SAs anchor to components of the extracellular matrix. Moreover, we show that ProCARs are intratumorally activated by probiotically-delivered SAs, receive further stimulation from bacterial TLR agonists, and are safe and effective in multiple xenograft models. This approach repurposes tumor-colonizing bacteria as beacons that guide the activity of engineered T cells, and in turn builds the foundation for communities of living medicines.

Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires well-characterized positive and negative control samples. Because appropriate tissue or cell line controls are not always available, a simple method to create synthetic IHC controls may be beneficial. Such a method is described here. It is adaptable to various antigen types, including proteins, peptides, or oligonucleotides, in a wide range of concentrations. This protocol explains the steps necessary to create synthetic antigen controls, using as an example a peptide from the human erythroblastic oncogene B2 (ERBB2/HER2) intracellular domain (ICD) recognized by a variety of diagnostically relevant antibodies. Serial dilutions of the HER2 ICD peptide in bovine serum albumin (BSA) solution are mixed with formaldehyde and heated for 10 min at 85 °C to solidify and cross-link the peptide/BSA mixture. The resulting gel can be processed, sectioned, and stained like a tissue, yielding a series of samples of known antigen concentrations spanning a wide range of staining intensities. This simple protocol is consistent with routine histology lab procedures. The method requires only that the user have a sufficient quantity of the desired antigen. Recombinant proteins, protein domains, or linear peptides that encode relevant epitopes may be synthesized locally or commercially. Laboratories generating in-house antibodies can reserve aliquots of the immunizing antigen as the synthetic control target. The opportunity to create well-defined positive controls across a wide range of concentrations allows users to assess intra- and inter-laboratory assay performance, gain insight into the dynamic range and linearity of their assays, and optimize assay conditions for their particular experimental goals.

Engineered outer membrane vesicles (OMVs) derived from laboratory strains of bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. As mimics of the bacterial cell surface, OMVs offer a molecularly-defined architecture for programming repetitive, high-density display of heterologous antigens in conformations that elicit strong B and T cell immune responses. However, antigen display on the surface of OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. To address this shortcoming, we created a universal approach called AddVax (avidin-based dock- and-display for vaccine antigen cross (x)-linking) whereby virtually any antigen that is amenable to biotinylation can be linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen receptor (SNARE) comprised of an outer membrane scaffold protein fused to a member of the avidin family. We show that SNARE-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations were injected in wild-type BALB/c mice, strong antigen-specific antibody responses were observed that depended on the physical coupling between the antigen and SNARE-OMV delivery vehicle. Overall, these results demonstrate AddVax as a modular platform for rapid self-assembly of antigen-studded OMVs with the potential to accelerate vaccine generation, respond rapidly to pathogen threats in humans and animals, and simplify vaccine stockpiling.

Introduction: In the Americas, Brazil contributes 91.63% of the total cases and the state of Para still has high endemia for leprosy. Objective: To analyze the performance of a rapid test for the diagnosis and epidemiological surveillance of leprosy in endemic areas. Methods: The sample consisted of 70 MB multibacillary leprosy (MB) patients, 63 paucibacillary (PB) patients, and 80 intradomiciliary consanguineous contacts (ICSCO) of patients. A rapid test with a 15-minute reading was applied using two prototypes: prototype 1, double test with trisaccharide antigen (NT-P-BSA) at 1a. line (83.2 ng/test) and disaccharide antigen (ND-O-BSA) at 2a. (83.2 ng/test), both with a flow of 0.08 μL/mm with a 10 μC membrane, anti-IgM conjugate with a flow of 0.040 μL/mm and a Tris-Triton and prototype 2 runner buffer with MIX antigen (trisaccharide + disaccharide) in the same concentrations and conditions of prototype 1. Results: The comparison of the MIX test positivity rate and the disaccharide or trisaccharide doublet test across all samples was statistically significant, demonstrating that the MIX test had higher seropositivity rates compared to the ND-O-BSA or NT-P-BSA. It was demonstrated that the MIX test showed a good performance, with 25.39% of the PB patients negative for the disaccharide and trisaccharide duplet test, but positive for MIX. Conclusions: These data suggest the potential for further optimizing the performance by adding other synthetic antigens to the MIX antigens.

Seven artificial mycobacterial antigens have been synthesized via the conjugation of synthetic carbohydrate epitopes of M. leprae phenolic glycolipid (PGL-1) and M. tuberculosis lipoarabinomannan (LAM) with bovine serum albumin (BSA). Each antigen has a different number of carbohydrate epitopes with a different structure and input sequence of the sugar residues. According to the serum reactivity, only three synthesized antigens can be useful for serodiagnosis of mycobacteriosis: (1) the antigen based on a disaccharide derivative of PGL-1: (DMG–Rha-AEP)-[BSA]; (2) the antigen based on a disaccharide fragment of PGL-1 and a hexasaccharide fragment of LAM: (DMG–Rha-AEP)-[BSA]–(Ara6-EG6); (3) the antigen based on the LAM diarabinofuranosyl fragment (Ara2-AEP)–[BSA].

The fully characterization of tumor associated antigens (TAAs) and of tumor associated carbohydrate antigens (TACAs) have opened the avenue of cancer immunotherapy. The intrinsic poor immunogenicity of TACAs, however, spotlighted the importance of multivalent presentation of the antigen(s) to trigger an immune response. Nanoparticles are excellent scaffolds for this purpose. Here we reported on the easy glycosylation of iron-based and biocompatible dextran-based nanoparticles with 1, a mimetic of the TnThr antigen. The multivalent presentation of 1 induced the induction of TNF-α and IL-6/IL10, respectively. The multivalent glycosylation of silica nanoparticles (GSiNPs) was also performed and saccharide loading qualitative assessed by solid state NMR. Our results offer the proof of concept that biomolecules coating can also be investigated on solid system by NMR.

Escherichia coli O157:H7 is considered as emerging foodborne pathogens that occur globally. Three major virulence protein factors; EspA(E), intimin(I), Tir(T) and Stx2 toxin have been found to be highly associated with bloody diarrhoea or, Haemolytic Uremic Syndrome. In this study, a trivalent recombinant EIT in combination with the binding domain of STX toxin were encapsulated with chitosan nanoparticles as a combination vaccine candidate. Mice were immunized either subcutaneously or orally with these antigens and challenged with E. coli O157:H7. Results of the binding inhibition assay with caco2 cell monolayer show a significant reduction in the adhesion percentage of pre-treated E. coli O157:H7 with immunized mice sera. Evaluation of neutralizing abilities of immune sera pre-incubated with CD50 dose of STX2 by Vero cells cytotoxicity neutralization assay shows less morphological reforms in comparison with the control groups. Results of mice mortality challenge with STX2 demonstrate around 66% of survived in immunized mice. In a challenge experiment with E. coli O157:H7, all the immunized mice showed a significant decrease in bacterial colonization and shedding. The results indicate that the use of multiple recombinant proteins in combination with natural nanostructure effectively evocated strong humoral and mucosal response, increasing the protection capacity of the synthetic antigen.

Hydatidosis is one of the most important diseases common between animals and human beings. Caused by Echinococcus granulosus tapeworm, the disease has a global epidemic. The serological diagnostic tests that are now utilized to confirm the imaging approaches have some drawbacks such as low sensitivity and cross-reaction with the serum of the patients infected with other parasites. The application of recombinant and synthetic antigens has proven improvement in the functionality of serological diagnostic tests. The purpose of this study was to demonstrate the expression and purification of truncated recombinant B8/1 (trB8/1) antigen and its application in ELISA for diagnosis of hydatid infection in human. The tEgB8/1 was colonized in the expression vector pET28b (+) and expressed in different strains of E. coli. This protein was purified by Ni2+-NTA chromatography. The antigenicity of the protein was evaluated by Western blotting and ELISA. In the test, 50 positive serum samples from hydatid infected patients, 50 samples from healthy people, and 30 serum samples from patients with other parasitic diseases were used to determine the sensitivity and the specificity of this antigen. The measured sensitivity and specificity of this antigen were identified to be 75.75% and 96.38% respectively. The P value of

The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by endowing the exchange of information between cells engaged in physical contacts. Beyond the integration of antigen (signal one), co-stimulation (signal two), and cytokines (signal three), the IS facilitates the delivery of T-cell effector assemblies including supramolecular attack particles (SMAPs) and extracellular vesicles (EVs). How these particulate outputs differ among T -cell subsets and how subcellular compartments and signals exchanged at the synapse contribute to their composition is not fully understood. Here we harnessed bead-supported lipid bilayers (BSLBs) as a tailorable and versatile technology for the study of synaptic particle biogenesis and composition in different T-cell subsets, including CART. These synthetic antigen-presenting cells (APCs) facilitated the characterisation of trans-synaptic vesicles (tSV) as a heterogeneous population of EVs comprising among others PM-derived synaptic ectosomes and CD63+ exosomes. We harnessed BSLB to unveil the factors influencing the vesicular release of CD40L, as a model effector, identifying CD40 trans presentation, T-cell activation, ESCRT upregulation/recruitment, antigen density/potency, co-repression by PD-1 ligands, and its processing by ADAM10 as major determinants. Further, BSLB made possible the comparison of microRNA (miR) species associated with tSV and steadily released EVs. Altogether, our data provide evidence for a higher specialisation of tSV which are enriched not only in effector immune receptors but also in miR and RNA-binding proteins. Considering the molecular uniqueness and functional complexity of the tSV output, which is also accompanied by SMAPs, we propose their classification as signal four.Graphical abstractHighlightsBead Supported Lipid Bilayers (BSLB) reconstituting antigen-presenting cells support synapse assembly by T cells and the release of effector particles.BSLB facilitate the dissection of the cellular machineries and synapse composition shaping the released tSV.tSV and their steadily released counterparts have a different composition. TSV show a higher enrichment of effectors including immune receptors, miR, RNA- and other nucleic acid-binding proteins, than EVs.

CD8(+) T cells express T cell receptors (TCRs) that recognize short peptide antigens in the context of major histocompatibility class I (MHC I) molecules. This recognition process produces an array of cytokine-mediated signals that help to govern immunological responses. Design of biostable MHC I peptide vaccines containing unnatural subunits is desirable, and synthetic antigens in which a native α-amino acid residue is replaced by a homologous β-amino acid residue (native side chain but extended backbone) might be useful in this regard. We have evaluated the impact of α-to-β backbone modification at a single site on T cell-mediated recognition of six clinically important viral and tumor-associated antigens bound to an MHC I. Effects of this modification on MHC I affinity and T cell activation were measured. Many of these modifications diminish or prevent T cell response. However, a number of α/β-peptide antigens were found to mimic the activity of natural antigens or to enhance maximal T cell response, as measured by interferon-γ release. Results from this broad exploratory study advance our understanding of immunological responses to antigens bearing unnatural modifications and suggest that α/β-peptides could be a source of potent and proteolytically stable variants of native antigens.

There is an urgent need for inexpensive, rapid and specific antigen-based assays to test for infection with SARS-CoV-2 and distinguish variants arising as the COVID-19 pandemic spreads. We have identified a small, synthetic protein (JS7), representing a region of maximum variability within the receptor binding domain (RBD), which binds antibodies in sera from nine patients with PCR-verified COVID-19 of varying severity. Antibodies binding to either JS7 or the SARS-CoV-2 recombinant RBD, as well as those that disrupt binding between a fragment of the ACE2 receptor and the RBD, are proportional to disease severity and clinical outcome. Binding to JS7 was inhibited by linear peptides from the RBD interface with ACE2. Variants of JS7, such as N501Y, can be quickly synthesized in a pure form in large quantities by automated methods. JS7 and related synthetic antigens can provide a basis for specific diagnostics for SARS-CoV-2 infections.

Natural populations of microbes and their hosts are engaged in an arms race in which microbes diversify to escape host immunity while hosts evolve novel immunity. This co-evolutionary process, known as the “Red Queen” hypothesis, poses a fundamental challenge to the development of broadly effective vaccines and diagnostics against a diversifying pathogen. Based on surveys of natural allele frequencies and experimental immunization of mice, we show minimal antigenic cross-reactivity among natural variants of the outer surface protein C (OspC), a dominant antigen of a Lyme Disease-causing bacterium (Borrelia burgdorferi). To overcome the challenge of OspC antigenic diversity to clinical development of preventive measures, we implemented a number of evolution-based strategies to broaden OspC immunological cross-reactivity. In particular, the centroid algorithm – a genetic algorithm to minimize sequence differences with natural variants – generated synthetic OspC analogs with the greatest promise as diagnostic and vaccine candidates against diverse Lyme pathogen strains coexisting in the Northeast United States. Mechanistically, we propose a model of runaway maximum antigen di-versification (MAD) mediated by amino-acid variations distributed across hypervariable regions on the OspC molecule. Under the MAD model, evolutionary centroids display high cross-reactivity by occupying the central void in the antigenic space excavated by diversifying natural variants. In contrast to the vaccine design based on concatenated epitopes, the centroid algorithm generates analogs of native antigens and is automated. The MAD model and evolution-inspired antigen designs have broad implications for combating diversifying pathogens driven by pathogen-host coevolution.ImportanceMicrobial pathogens rely on molecular diversity of cell surface antigens to escape host immunity. Vaccines based on one antigen variant often fail to protect the host against pathogens carrying other variants. Here we show evolution-based designs of synthetic antigens that are broadly reactive to all natural variants. The evolutionary analogs of a major surface antigen of a Lyme disease bacterium (Borrelia burgdorferi) showed promise as vaccine candidates against diverse pathogen strains coexisting in the endemic areas of Lyme disease in Northeast United States. Our evolution-based computational design is automated, generates molecular analogs of natural antigens, and opens a novel path to combating fast-evolving microbial pathogens.

Most rheumatic diseases, including rheumatoid arthritis (RA), are characterized by immune disorders that affect antibody activity. In the present study, using Dot blot and ELISA assay, we showed that patients with rheumatic disease produced significantly more antibodies against lipopolysaccharide (LPS) P. mirabilis O3 compared to healthy donors (p &lt, 0.05), and affinity purified antibodies against LPS O3 may cross-react with collagen type I. It was demonstrated that purified of antibodies isolated from RA patients sera, reacted stronger with the collagen than healthy donors (p = 0.015), and cross-reaction was correlated with level of anti-citrullinated peptide antibodies (r = 0.7, p = 0.003). Moreover, using six different lipopolysaccharides were demonstrated the significant correlations in sera reactivity among lysine-containing lipopolysaccharides observed in patients&rsquo, sera (p &lt, 0.05). Using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) it was shown that unique wavenumbers of sera spectra correlate with reactivity with lipopolysaccharides allowing distinguish patients from healthy blood donors. Antibodies adsorption by synthetic antigens shows that in patients&rsquo, group anti-LPS O3 antibodies can be adsorbed by both amides of galacturonic acid and lysine or threonine, which suggests less specificity of antibodies binding with non-carbohydrate LPS component. The observed correlations suggest that non-carbohydrate components of LPS may be an important epitope for less specific anti-LPS antibodies, which might lead to cross-reactions and affect disease development.

Over the last four decades, significant efforts have been invested to develop vaccines against malaria. Although most efforts are focused on the development of; P. falciparum; vaccines, the current availability of the parasite genomes, bioinformatics tools, and high throughput systems for both recombinant and synthetic antigen production have helped to accelerate vaccine development against the; P. vivax; parasite. We have previously; in silico; identified several; P. falciparum; and; P. vivax; proteins containing α-helical coiled-coil motifs that represent novel putative antigens for vaccine development since they are highly immunogenic and have been associated with protection in many; in vitro; functional assays. Here, we selected five pairs of; P. falciparum; and; P. vivax; orthologous peptides to assess their sero-reactivity using plasma samples collected in; P. falciparum-; endemic African countries.; Pf-Pv; cross-reactivity was also investigated. The pairs; Pf27/Pv27, Pf43/Pv43; , and; Pf45/Pv45; resulted to be the most promising candidates for a cross-protective vaccine because they showed a high degree of recognition in direct and competition ELISA assays and cross-reactivity with their respective ortholog. The recognition of; P. vivax; peptides by plasma of; P. falciparum; infected individuals indicates the existence of a high degree of cross-reactivity between these two; Plasmodium; species. The design of longer polypeptides combining these epitopes will allow the assessment of their immunogenicity and protective efficacy in animal models.

With nontuberculous mycobacterial pulmonary disease (NTM-PD) increasing in prevalence worldwide, there is a pressing clinical need for improved diagnostic techniques. There are several problems with the current diagnostic framework, requiring positive microbiology, supportive clinical features and characteristic radiological findings. Serology has previously been investigated as an adjunct to diagnosis of NTM-PD in several pilot studies suggesting potential benefit. We performed a case-control study investigating the diagnostic accuracy of a novel ELISA assay using synthetic antigens based on single mycolic acids, which are ubiquitous cell wall components of mycobacteria, in the diagnosis of NTM-PD. Adult patients with either cystic fibrosis or bronchiectasis were recruited, and demographic data including details of NTM-PD and previous treatment were recorded. Serum samples were taken on a single occasion from each subject. Each sample was tested against an array of antigens. Overall, 62 patients with cystic fibrosis and 29 patients with bronchiectasis of other causes were recruited, including 25 patients with NTM-PD due to M. abscessus, 17 with NTM-PD due to Mycobacterium avium complex (MAC), 9 patients with possible NTM-PD, and 40 disease controls with either cystic fibrosis or bronchiectasis. ELISA testing was variably positive to 25 individual antigens, with a sensitivity up to 88% for the M. abscessus patients and 68% for the MAC group. Specificity ranged from 77.5-90%. The results of this study suggest that serology shows promise as an adjunct to current diagnostic methods in NTM-PD. Further studies in a larger, prospective cohort are needed.

Vaccines based on isolated polysaccharides successfully protect humans from bacterial pathogens such as Streptococcus pneumoniae. Because polysaccharide production and isolation can be technically challenging, glycoconjugates containing synthetic antigens are an attractive alternative. Typically, the shortest possible oligosaccharide antigen is preferable as syntheses of longer structures are more difficult and time-consuming. Combining several protective epitopes or polysaccharide repeating units as blocks by bonds other than glycosidic linkages would greatly reduce the synthetic effort if the immunological response to the polysaccharide could be retained. To explore this concept, we bridged the well-understood and immunologically potent RU of S. pneumoniae serotype 14 (ST14) with an aliphatic spacer and conjugated it to the carrier protein CRM197. Mice immunized with the spacer-bridged glycan conjugates produced high levels of specific antibodies after just one or two vaccine doses, while the tetrasaccharide repeating unit alone required three doses. The antibodies recognized specifically ST14 CPS, while no significant antibody levels were raised against the spacer or unrelated CPS. Synthetic vaccines generated antibodies with opsonic activity. Mimicking polysaccharides by coupling repeating unit antigens via an aliphatic spacer may prove useful also for the development of other glycoconjugate vaccine candidates, thereby reducing the synthetic complexity while enhancing a faster immune response.

Synthetic antigens based on consensus sequences that represent circulating viral isolates are sensitive, time saving and cost-effective tools for in vitro immune monitoring and to guide immunogen design. When based on a representative sequence database, such consensus sequences can effectively be used to test immune responses in exposed and infected individuals at the population level. To accelerate immune studies in SARS-CoV-2 infection, we here describe a SARS-CoV-2 2020 consensus sequence (CoV-2-cons) which is based on more than 1700 viral genome entries in NCBI and encompasses all described SARS-CoV-2 open reading frames (ORF), including recently described frame-shifted and length variant ORF. Based on these sequences, we created curated overlapping peptide (OLP) lists containing between 1500 to 3000 peptides of 15 and 18 amino acids in length, overlapping by 10 or 11 residues, as ideal tools for the assessment of SARS-CoV-2-specific T cell immunity. In addition, CoV-2-cons sequence entropy values are presented along with variant sequences to provide increased coverage of the most variable sections of the viral genome. The identification of conserved protein fragments across the coronavirus family and the corresponding OLP facilitate the identification of T cells potentially cross-reactive with related viruses. This new CoV-2-cons sequence, together with the peptides sets, should provide the basis for SARS-CoV-2 antigen synthesis to facilitate comparability between ex-vivo immune analyses and help to accelerate research on SARS-CoV-2 immunity and vaccine development.

Vaccines have shown significant promise in eliciting protective and therapeutic responses. However, most effective vaccines require several booster shots, and it is challenging to generate potent responses against small molecules and synthetic peptide antigens often used to increase target specificity and improve vaccine stability. As continuous antigen uptake and processing by APCs and persistent toll-like receptor (TLR) priming have been shown to amplify antigen specific humoral immunity, we explored whether a single injection of a mesoporous silica micro-rod (MSR) vaccine containing synthetic molecules and peptides can effectively generate potent and durable antigen-specific humoral immunity. A single injection of the MSR vaccine against a gonadotropin-releasing hormone (GnRH) decapeptide elicited highly potent anti-GnRH response that lasted for over 12 months. The MSR vaccine generated higher titers than bolus or alhydrogel alum vaccine formulations. Moreover, a MSR vaccine directed against a Her2/neu peptide within the Trastuzumab binding domain showed immunoreactivity to native Her2 protein on tumor cell surface and, when directed against nicotine, generated long-term anti-nicotine antibodies. Mechanistically, we found that the MSR vaccine induced persistent germinal center (GC) B-cell activity for more than 3 weeks after a single injection, generation of memory B cells, and that at least 7 days of immunostimulation by the vaccine was required to generate an effective humoral response. Together, these data suggest that the MSR vaccine represents a promising technology for synthetic antigen vaccines to bypass the need for multiple immunizations and enhance long-term production of antibodies against endogenous antigens in the context of reproductive biology, cancer, and chronic addiction.

The current vaccine for tuberculosis (TB) is a live attenuated strain of Mycobacterium bovis (BCG) and while effective at reducing the potential for disseminated TB in young children its disease protection rates in adults is highly variable while it confers little protection against latent TB. With these limitations a new vaccine is desperately needed. We investigated the efficacy of three members of the mycobacterial membrane protein Large (MmpL) family as potential subunit vaccines for TB. MmpLs are large, multifunctional integral membrane proteins, and as such are recalcitrant to purification. Here, we describe a strategy of producing synthetic antigens comprised of the soluble, extracellular regions of MmpL (SERoM)-1, MmpL8 and MmpL10 (SERoM-8 and 10 respectively) as potential vaccine candidates. SERoM-1 and SERoM-8 were determined to be highly immunogenic by IFN-γ ELISpot assays, with 0.1% of all splenocytes from SERoM-1 vaccinated mice producing IFN-γ when re-stimulated with MmpL1. A combined SERoM-1, −8 and −10 vaccine demonstrated significant protection against M. tuberculosis challenge in a murine model of TB, resulting in approximately 10-fold reduction in bacterial numbers following challenge in both the lungs and spleens compared to adjuvant only vaccinated mice. These protective effects were comparable to that achieved with BCG.

The first glycoconjugate vaccine using isolated glycans was licensed to protect children from Haemophilus influenzae serotype b (Hib) infections. Subsequently, the first semisynthetic glycoconjugate vaccine using a mixture of antigens derived by polymerization targeted the same pathogen. Still, a detailed understanding concerning the correlation between oligosaccharide chain length and the immune response towards the polyribosyl-ribitol-phosphate (PRP) capsular polysaccharide that surrounds Hib remains elusive. The design of semisynthetic and synthetic Hib vaccines critically depends on the identification of the minimally protective epitope. Here, we demonstrate that an octasaccharide antigen containing four repeating disaccharide units resembles PRP polysaccharide in terms of immunogenicity and recognition by anti-Hib antibodies. Key to this discovery was the development of a modular synthesis that enabled access to oligosaccharides up to decamers. Glycan arrays containing the synthetic oligosaccharides were used to analyze anti-PRP sera for antibodies. Conjugates of the synthetic antigens and the carrier protein CRM197, which is used in licensed vaccines, were employed in immunization studies in rabbits.

There is an urgent need for inexpensive, rapid and specific antigen-based assays to test for vaccine efficacy and detect infection with SARS-CoV-2 and its variants. We have identified a small, synthetic protein (JS7), representing a region of maximum variability within the receptor binding domain (RBD), which binds antibodies in sera from nine patients with PCR-verified COVID-19 of varying severity. Antibodies binding to either JS7 or the SARS-CoV-2 recombinant RBD, as well as those that disrupt binding between a fragment of the ACE2 receptor and the RBD, are proportional to disease severity and clinical outcome. Binding to JS7 was inhibited by linear peptides from the RBD interface with ACE2. Variants of JS7, such as E484 K or N501Y, can be quickly synthesized in a pure form in large quantities by automated methods. JS7 and related synthetic antigens can provide a basis for specific diagnostics for SARS-CoV-2 infections.

The available cystic echinococcosis immunodiagnostic tools cannot assess cyst viability. Here, synthetic antigen B (AgB) subunits were structurally and immunologically characterized. They all showed propensity to self-oligomerize, similarly to native AgB, and AgB1 held promise as a marker of viability., Background Several tools have been proposed for serodiagnosis of cystic echinococcosis (CE), but none seems promising for cyst viability assessment. Antigens with stage-specific diagnostic value have been described, but few studies with well-characterized antigens and human serum samples have been performed. Antigen B (AgB) proteoforms hold promise as markers of viability, due to their differential stage-related expression and immunoreactivity. Methods Four AgB subunits (AgB1, AgB2, AgB3, AgB4) were synthesized and structurally characterized. Based on the preliminary evaluation of the subunits by western immunoblotting and enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples. Results All subunits showed a high degree of spontaneous oligomerization. Interacting residues within oligomers were identified, showing that both the N-terminal and C-terminal of each subunit are involved in homo-oligomer contact interfaces. No hetero-oligomer was identified. AgB1 and AgB2 ELISAs revealed different sensitivities relative to cyst stage. Of note, besides high specificity (97.2%), AgB1 revealed a higher sensitivity for active-transitional cysts (100% for CE1, 77.8% for CE2, 81.5% for CE3a, and 86.3% for CE3b) than for inactive cysts (41.7% for CE4 and 11.1% for CE5) and postsurgical patients (44%). Interestingly, 19 of 20 patients with spontaneously inactive cysts and 6 of 9 treated with albendazole >5 years earlier were negative on the AgB1 assay. Conclusions The structural characterization of subunits provides insights into the synthetic antigen conformation. The stage-related sensitivity of synthetic AgB1 holds promise as part of a multiantigen setting and deserves further longitudinal evaluation as marker of cyst viability.

Mucosal-associated invariant T (MAIT) cells are activated by unstable antigens formed by reactions of 5-amino-6-D-ribitylaminouracil (a vitamin B2 biosynthetic intermediate) with glycolysis metabolites such as methylglyoxal. Here we show superior preparations of antigens in dimethylsulfoxide, avoiding their rapid decomposition in water (t1/2 1.5 h, 37 °C). Antigen solution structures, MAIT cell activation potencies (EC50 3–500 pM), and chemical stabilities are described. Computer analyses of antigen structures reveal stereochemical and energetic influences on MAIT cell activation, enabling design of a water stable synthetic antigen (EC50 2 nM). Like native antigens, this antigen preparation induces MR1 refolding and upregulates surface expression of human MR1, forms MR1 tetramers that detect MAIT cells in human PBMCs, and stimulates cytokine expression (IFNγ, TNF) by human MAIT cells. These antigens also induce MAIT cell accumulation in mouse lungs after administration with a co-stimulant. These chemical and immunological findings provide new insights into antigen properties and MAIT cell activation., MAIT cells are activated by MR1 restricted antigens derived from riboflavin biosynthesis. Here the authors characterize MAIT cell antigenicity and synthesize a water stable antigen that activates human MAIT cells in vitro and mouse MAIT cells in vivo.

Background & Aim Clinical success of engineered T cell therapeutics against blood cancers has fueled rapid evolution of the field of T cell-based therapies and created demand for new antigen-binding domains. Among different tumor-targeting domains, single-chain antibodies (scFvs) remain the most prevalent. Extensive screening of scFvs for unwanted cross-reactivities with healthy tissues can reduce the likelihood of selecting ligands reactive to host tissues but not completely eliminate the possibility. Here we describe our experience with two BCMA-specific scFvs and the effect of non-toxic tissue cross-reactivity on the performance of engineered T cells. Methods, Results & Conclusion BCMA-specific scFvs derived from the monoclonal antibodies C11D5.3 and J22.9-xi were used as antigen-binding domains on two synthetic antigen receptor frameworks, T cell antigen coupler (TAC) receptor and chimeric antigen receptor (CAR), and tested against multiple myeloma xenografts. When T cells were engineered with the TAC framework, both scFvs yielded TAC T cells with equivalent in vitro performance; however, the J22.9-xi TAC T cells were more therapeutically robust in vivo. Further analysis revealed superior in vivo expansion of J22.9-xi TAC T cells, compared to C11D5.3 TAC T cells. Surprisingly, J22.9-xi TAC T cells expanded equally well in tumor-free and tumor-bearing mice in the absence of toxicity, suggesting that cross-reactivity to the host supported in vivo expansion and efficacy of J22.9-xi TAC T cells. In the context of a CD28-based CAR, T cells engineered with the J22.9-xi CAR displayed markedly greater levels of exhaustion, compared with T cells engineered with the C11D5.3 CAR, as evidenced by upregulation of checkpoint receptors, higher basal cytokine production, and reduced ability to upregulate cytokine production upon stimulation. Despite functional in vitro impairments, the J22.9-xi-28ζ CAR T cells proved to be the most efficacious and persistent in vivo when compared to the other engineered T cell populations; again, therapeutic efficacy was not associated with overt toxicities. Taken together, these observations suggest that non-lethal cross-reactivity of engineered T cells can augment in vivo potency and enable therapeutic efficacy of T cell products with evidence of functional exhaustion.

Streptococcus pneumoniae 19A (ST19A) and 19F (ST19F) are among the prevalent serotypes causing pneumococcal disease worldwide even after introduction of a 13-valent pneumococcal conjugate vaccine (PCV13). Synthetic glycoconjugate vaccines have defined chemical structures rather than isolated polysaccharide mixtures utilized in marketed vaccines. Ideally, a minimal number of synthetic antigens would cover as many bacterial serotypes to lower cost of goods and minimize the response to carrier proteins. To demonstrate that a chimeric oligosaccharide antigen can induce a protective immune response against multiple serotypes, we synthesized a chimeric antigen (ST19AF) that is comprised of a repeating unit of ST19A and ST19F capsular polysaccharide each. Synthetic glycan epitopes representing only ST19A, and ST19F were prepared for comparison. Semisynthetic glycoconjugates containing chimeric antigen ST19AF induced high antibody titers able to recognize native CPS from ST19A and ST19F in rabbits. The antibodies were able to kill both strains of pneumococci. Chimeric antigens are an attractive means to induce an immune response against multiple bacterial serotypes.

Neuropsychiatric systemic lupus erythematosus is an autoimmune disorder characterized by an irregular exchange between the central nervous system and the immune system, leading to the outbreak of neurological conditions with possible disabling effects. Although neuropsychiatric systemic lupus erythematosus is the most common expression of lupus condition, it is still poorly understood. In this study, we focus on the development of an advantageous method based on the application of synthetic nucleic acids and protein-based antigen arrays in order to characterize autoreactive antibodies in neuropsychiatric systemic lupus erythematosus. We confirmed the benefits of using synthetic oligonucleotides such as assay reproducibility, elevated affinity and specificity to autoreactive antibodies. We also demonstrated presence of autoantibodies towards three particular synthetic double stranded antigens and verify similarity of antinuclear antibody patterns in ordinary lupus and neuropsychiatric systemic lupus erythematosus., Highlights • Neuropsychiatric systemic lupus erythematosus is the most common expression of lupus condition. • Neuropsychiatric systemic lupus erythematosus is still poorly understood. • Antibodies to synthetic nucleic acid antigens differentiate systemic lupus erythematosus. • Autoantibody levels in NPSLE correlate with the disease activity but not with the organic brain dysfunction.

UID/Multi/04378/2019. POCI-01-0145-FEDER-007728. UID/QUI/50006/2019. POCI-01-0145-FEDER-007265. SFRH/BD/108433/2015. GG/001/2014. Pneumocystis jirovecii pneumonia (PcP) is a major human immunodeficiency virus (HIV)-related illness, rising among immunocompromised non-HIV patients and in developing countries. Presently, the diagnosis requires respiratory specimens obtained through invasive and costly techniques that are difficult to perform in all patients or implement in all economic settings. Therefore, the development of a faster, cost-effective, non-invasive and field-friendly test to diagnose PcP would be a significant advance. In this study, recombinant synthetic antigens (RSA) of P. jirovecii’s major surface glycoprotein (Msg) and kexin-like serine protease (Kex1) were produced and purified. These RSA were applied as antigenic tools in immunoenzymatic assays for detection of specific anti-P. jirovecii antibodies (IgG and IgM) in sera of patients with (n = 48) and without (n = 28) PcP. Results showed that only IgM anti-P. jirovecii levels were significantly increased in patients with PcP compared with patients without P. jirovecii infection (p ≤ 0.001 with both RSA). Thus, two strip lateral flow immunoassays (LFIA), based on the detection of specific IgM anti-P. jirovecii antibodies in human sera samples, were developed using the innovative association of P. jirovecii’s RSA with spherical gold nanoparticles (AuNPs). For that, alkanethiol-functionalized spherical AuNPs with ca. ~40 nm in diameter were synthetized and conjugated with the two RSA (Msg or Kex1) produced. These AuNP-RSA conjugates were characterized by agarose gel electrophoresis (AGE) and optimized to improve their ability to interact specifically with serum IgM anti-P. jirovecii antibodies. Finally, two LFIA prototypes were developed and tested with pools of sera from patients with (positive sample) and without (negative sample) PcP. Both LFIA had the expected performance, namely, the presence of a test and control red colored lines with the positive sample, and only a control red colored line with the negative sample. These results provide valuable insights into the possibility of PcP serodiagnosis at point-of-care. The optimization, validation and implementation of this strip-based approach may help to reduce the high cost of medical diagnosis and subsequent treatment of PcP both in industrialized and low-income regions, helping to manage the disease all around the world. publishersversion published

Chimeric antigen receptor T (CAR-T) cells are T cells engineered to express specific synthetic antigen receptors that can recognize antigens expressed by tumor cells, which after the binding of these antigens to the receptors are eliminated, and have been adopted to treat several kinds of malignancies. Autoimmune diseases (AIDs), a class of chronic disease conditions, can be broadly separated into autoantibody-mediated and T cell-mediated diseases. Treatments for AIDs are focused on restoring immune tolerance. However, current treatments have little effect on immune tolerance inverse; even the molecular target biologics like anti-TNFα inhibitors can only mildly restore immune balance. By using the idea of CAR-T cell treatment in tumors, CAR-T cell-derived immunotherapies, chimeric autoantibody receptor T (CAAR-T) cells, and CAR regulatory T (CAR-T) cells bring new hope of treatment choice for AIDs.

The complicated clinical picture and biomolecular pattern of human autoimmune diseases (ADs) make knowledge on their etiology still fragmentary. The diagnostic approaches for ADs require improvement both for clinical and research effort to progress. Synthetic biomolecular antigens find growing applications for diagnosis and investigation of ADs. The main goal of this work is to detect interaction between synthetic antigens and autoantibodies in systemic lupus erythematosus within a combined, high-throughput assay. A panel of synthetic antigens has been prepared from DNA, RNA, locked nucleic acids and apolipoprotein H. The binding of synthetic antigens to autoantibodies has been confirmed in sera samples from those with active systemic lupus erythematosus (SLE) by indirect enzyme linked immunosorbent assay. Our study provides an efficient methodology for combined autoantibody profiling in SLE.

Subunit vaccines can have excellent safety profiles, but their ability to give rise to robust immune responses is often compromised. For glycan-based vaccines, insufficient understanding of B and T cell epitope combinations that yield optimal immune activation hinders optimization. To determine which antigen features promote desired IgG responses, we synthesized epitope-functionalized polymers using ring-opening metathesis polymerization (ROMP) and assessed the effect of B and T cell epitope loading. The most robust responses were induced by polymers with a high valency of B and T cell epitopes. Additionally, IgG responses were greater for polymers with T cell epitopes that are readily liberated upon endosomal processing. Combining these criteria, we used ROMP to generate a non-toxic, polymeric antigen that elicited stronger antibody responses than a comparable protein conjugate. These findings highlight principles for designing synthetic antigens that elicit strong IgG responses against inherently weak immune targets such as glycans.

Background: The search for a tissue-mass reducing reproductive hormone involved a bioassay-guided physicochemical fractionation of sheep blood plasma. This brought forth a candidate protein whose apparent mass on gels and in mass spectrometry (MS) was 7-8 kDa, implying a polypeptide of ~70 residues. Four purification runs gave Edman N-terminal sequences relating to 1MKPLTGKVKEFNNI14. This is bioinformatically obscure and has been resistant to molecular biological investigation. The sequence was synthesized as the peptide EPL001, against which was raised a goat polyclonal antiserum, G530. Used in an antigen capture campaign, G530 pointed to the existence of a novel derivative of secretogranin II (SgII), the neuroendocrine secretory vesicle helper protein and prohormone. The proposed SgII derivative was dubbed SgII-70, yet the sequence commonality between SgII and EPL001 is essentially NNI. Methods: Immunohistochemical (IHC) labelling with G530 is reported within rat, mouse and human cerebrovasculature and in glandular elements of the mouse intestine. Epitope mapping involved IHC peptide preabsorption, allied to deductive bioinformatics and molecular modelling in silico. Results: G530 is deemed monoepitopic in regard to both its synthetic antigen (EPL001) and its putative endogenous antigen (SgII related). The epitope within EPL001 of the anti-EPL001 antibody is inferred to be the contiguous C-terminal 9KEFNNI14. This is so because the G530 blockade data are consistent with the epitope in the mammalian endogenous antigen being part contiguous, part non-contiguous KE·F·NNI, ex hypothesi. The observed immunostaining is deduced to be due to pre-SgII-70, which has a non-C-terminal NNI, and SgII-70, which has an N-terminal MLKTGEKPV/N and a C-terminal NNI (these two motifs being in the reverse order in the SgII parent protein). Conclusion: The present data are consistent with the hypothesis that the anti-EPL001 antibody binds to an SgII-related epitope. SgII is apparently subject to peptide splicing, as has been reported for the related chromogranin A.

Thrombospondin-related adhesive protein (TRAP) is essential for sporozoite motility and the invasion of mosquitoes' salivary gland and vertebrate's hepatocyte and is, thus, considered a promising pre-erythrocytic vaccine candidate. Despite the existence of a few reports on naturally acquired immune response against Plasmodium vivax TRAP (PvTRAP), it has never been explored so far in the Amazon region, so results are conflicting. Here, we characterized the (IgG and IgG subclass) antibody reactivity against recombinant PvTRAP in a cross-sectional study of 299 individuals exposed to malaria infection in three municipalities (Cruzeiro do Sul, Mâncio Lima and Guajará) from the Acre state of the Brazilian Amazon. In addition, the full PvTRAP sequence was screened for B-cell epitopes using in silico and in vitro approaches. Firstly, we confirmed that PvTRAP is naturally immunogenic in the cohort population since 49% of the individuals were IgG-responders to it. The observed immune responses were mainly driven by cytophilic IgG1 over all other sublcasses and the IgG levels that was corelated with age and time of residence in the studied area (p < 0.05). Interestingly, only the levels of specific anti-TRAP IgG3 seemed to be associated with protection, as IgG3 responders presented a significantly higher time elapse since the last malaria episode than those recorded for IgG3 non-responders. Regarding the B-cell epitope mapping, among the 148 responders to PvTRAP, four predicted epitopes were confirmed by recognition of antibodies (PvTRAPR197−H227; PvTRAPE237−T258; PvTRAPP344−G374; and PvTRAPE439−K454). Nevertheless, the frequency of responders against these peptides were low and did not show a clear correlation with the antibody response against the corresponding antigen. Moreover, none of the linear confirmed epitopes were located in the binding regions of PvTRAP in respect to the host cell ligand. Collectively, our data confirm the PvTRAP immunogenicity among Amazon inhabitants, while suggesting that the main important B-cell epitopes are not linear.

The zwitterionic Streptococcus pneumoniae serotype 1 polysaccharide (Sp1) is an important anchor point for our immune system to act against streptococcal infections. Antibodies can recognize Sp1 saccharides, and it has been postulated that Sp1 can elicit a T-cell-dependent immune reaction as it can be presented by MHC-II molecules. To unravel the molecular mode of action of this unique polysaccharide we here describe the chemical synthesis of a set of Sp1 fragments, ranging from 3 to 12 monosaccharides in length. We outline a unique synthetic approach to overcome the major synthetic challenges associated with the complex Sp1 structure and provide a stereoselective route of synthesis for the oligosaccharide backbone as well as a strategy to introduce the carboxylic acid functions. Molecular dynamics (MD) simulations together with NMR spectroscopy studies reveal that the oligosaccharides take up helical structures with the nona- and dodecasaccharide completing a full helical turn. The 3D structure of the oligosaccharides coincides with the topology required for good interaction with anti-Sp1 antibodies, which has been mapped in detail using STD-NMR. Our study has revealed the Sp1 nona- and dodecasaccharides as promising synthetic antigens, displaying all (3D) structural elements required to mimic the natural polysaccharide and required to unravel the molecular mode of action of these unique zwitterionic polysaccharides., A stereoselective synthesis provides zwitterionic Streptococcus pneumoniae Sp1 oligosaccharides up to the 12-mer level, which adopt a helical structure, crucial for antibody interaction.

In the last two decades, the paramount importance of Tumor Associated Carbohydrate Antigens (TACAs) as targets for anticancer vaccine development has been firmly assessed. The Tn antigen is an ideal target for immunotherapy, in that it is masked on normal cells, but exposed on cancer cells. However, it is difficult to elicit an effective and long-lasting response against Tn antigen and other TACAs. Here we report on the Tn antigen analogues developed to boost the latent Tn immune response. Hopefully, the results reported herein will be of help for the rational design of effective TACA-based immunostimulants.

Optimization and standardization of immunohistochemistry (IHC) protocols within and between laboratories requires reproducible positive and negative control samples. In many situations, suitable tissue or cell line controls are not available. We demonstrate here a method to incorporate target antigens into synthetic protein gels that can serve as IHC controls. The method can use peptides, protein domains, or whole proteins as antigens, and is compatible with a variety of fixation protocols. The resulting gels can be used to create tissue microarrays (TMAs) with a range of antigen concentrations that can be used to objectively quantify and calibrate chromogenic, fluorescent, or mass spectrometry-based IHC protocols. The method offers an opportunity to objectively quantify IHC staining results, and to optimize and standardize IHC protocols within and between laboratories. (J Histochem Cytochem 58:XXX–XXX, 2019)

Adoptive T cell therapies are transforming the treatment of solid and liquid tumors, yet their widespread adoption is limited in part by the challenge of generating functional cells. T cell activation and expansion using conventional antigen-presenting cells (APCs) is unreliable due to the variable quality of donor-derived APCs. As a result, engineered approaches using nanomaterials presenting T cell activation signals are a promising alternative due to their ability to be robustly manufactured with precise control over stimulation cues. In this work, we design synthetic APCs that consist of liposomes surface-functionalized with peptide-major histocompatibility complexes (pMHC). Synthetic APCs selectively target and activate antigen-specific T cell populations to levels similar to conventional protocols using non-specific αCD3 and αCD28 antibodies without the need for costimulation signals. T cells treated with synthetic APCs produce effector cytokines and demonstrate cytotoxic activity when co-cultured with tumor cells presenting target antigenin vitro. Following adoptive transfer into tumor-bearing mice, activated cells control tumor growth and improve overall survival compared to untreated mice. Synthetic APCs could potentially be used in the future to improve the accessibility of adoptive T cell therapies by removing the need for conventional APCs during manufacturing.

This review describes the developments in the synthesis of teichoic acids (TA) - glycosylated poly(alditolphosphates) - and the application of these fragments in immunological studies. These structurally diverse biopolymers are omnipresent constituents of the Gram-positive bacterial cell wall where they fulfill a variety of vital functions. They have been and continue to be attractive synthetic targets because of their challenging structures and the fact that their microheterogeneity precludes their isolation in single and pure enough form from natural sources. Progress in glycosylation chemistry and the development of effective phosphorylation chemistry has driven TA synthesis over the years, and highly complex and large TA structures can now reliably be targeted. Starting from the first TA synthesis in 1981, this review highlights the progress made in the field over the years. The synthesized TA fragments have been used to unravel their role in immunology and it is described how focused libraries of TAs have been used to discover the active principles of the TA polymers that interact with the innate immune system. Recently, synthetic TA fragments have also found applications as well-defined synthetic antigens for the generation of novel vaccine modalities to combat Gram-positive bacterial infections. It is foreseen that synthetic TA fragments will be valuable tools in the future to unravel the mode of action of these biomolecules at the molecular level. They will be instrumental in discovering and characterizing their designated biological binding partners, be it pattern recognition receptors or carbohydrate binding lectins or biomachinery enzymes. This review thus serves to showcase the potential of organic synthesis for (chemical) biology and immunology.