Search Results (121)

Glycoconjugate vaccines, consisting of a protein component covalently linked to a glycan antigen, have led to a significant reduction in the global occurrence of bacterial meningitis and pneumonia. They provide robust, lasting immunity in all age groups. However, their production by traditional chemical conjugation approaches has drawbacks in terms of complexity, cost, and lack of flexibility in design, which explains their limited application to a few pathogenic bacteria in the past four decades. Protein glycan coupling technology (PGCT) or bioconjugation, where glycoconjugates are produced in purpose-engineered bacterial cells, is a useful alternative to chemical conjugation and promises an array of low-cost custom-made glycoconjugate vaccines with vast protein glycan combinations. The technology has undergone significant development since its inception, and new advances and refinements continually drive the field forward. Several bioconjugate vaccines are currently in clinical trials, demonstrating the potential of the technology. We will review the wide applicability of bioconjugation and recent developments in each of the components of the technology, namely, glycan expression, protein selection, and the coupling of selected glycan with proteins, all within custom-designed E. coli cells. These advances promise to deliver effective glycoconjugate vaccines for multiple unmet medical needs. Full article

Glycans on the surface of all immune cells are the product of diverse post-translational modifications (glycosylation) that affect almost all proteins and possess enormous structural heterogeneity. Their bioinformational content is decoded by glycan-binding proteins (lectins, GBPs), such as C-type lectins, including selectins, galectins, and Siglecs. Glycans located on the surface of immune cells are involved in many immunological processes through interactions with GBPs. Lectins recognize changes in the glycan epitopes; distinguish among host (self), microbial (non-self), and tumor (modified self) antigens; and consequently regulate immune responses. Understanding GBP–glycan interactions accelerates the development of glycan-targeted therapeutics in severe diseases, including inflammatory and autoimmune diseases and cancer. This review will discuss N- and O-glycosylations and glycosyltransferases involved in the biosynthesis of carbohydrate epitopes and address how interactions between glycan epitopes and GBPs are crucial in immune responses. The pivotal role of the glycan antigen tetrasaccharide sialyl Lewis x in mediating immune and tumor cell trafficking into the extravascular site will be discussed. Next, the role of glycans in modulating bacterial, fungal, viral, and parasitic infections and cancer will be surveyed. Finally, the role of glycosylation in antibodies and carbohydrate vaccines will be analyzed. Full article

We investigated HIV-1 immune evasion mechanisms in a slow progressor (CBJC515) by constructing pseudoviruses expressing autologous Env proteins. Intriguingly, all pseudoviruses exhibited resistance to the broadly neutralizing antibody (bNAb) PGT135. Using site-directed mutagenesis and chimeric Env construction, we identified distinct escape mechanisms: early 2005 strains lost the N332 glycan site, while 2006/2008 strains retained key epitopes but developed resistance through structural modifications in the V1/V4/C2 regions or acquired novel N-glycosylation sites (N398/N611). These findings provide insights into how HIV-1 can escape from N332-directed bNAb responses without altering the epitope itself. Furthermore, chimeric experiments also elucidated regional co-evolution and functional maintenance: the V1V2 region broadly interfered with envelope protein function, while the V3 region may exhibit compensatory activity, restoring functionality and mitigating deleterious polymorphisms in other regions to keep Env antigenic diversity. These results offer valuable mechanistic clues that may inform the development of next-generation HIV-1 vaccines. Full article

Background/Objectives: Rising antibiotic resistance underscores the urgent need for effective vaccines against shigellosis. Our previous research identified the Shigella flexneri 2a truncated mutant (STM), a wzy gene knock-out strain cultivated in shake-flasks, as a promising broadly protective Shigella vaccine candidate. Expanding on this finding, our current study explores the feasibility of transitioning to a fermentor-grown STM as a vaccine candidate for further clinical development. Methods: The STM and STM-Cj, engineered to express the conserved Campylobacter jejuni N-glycan antigen, were grown in animal-free media, inactivated with formalin, and evaluated for key antigen retention and immunogenicity in mice. Results: The fermentor-grown STM exhibited significantly increased production yields and retained key antigens after inactivation. Immunization with the STM, particularly along with the double-mutant labile toxin (dmLT) adjuvant, induced robust immune responses to the conserved proteins IpaB, IpaC, and PSSP-1. Additionally, it provided protection against homologous and heterologous Shigella challenges in a mouse pulmonary model. The STM-Cj vaccine elicited antibody responses specific to the N-glycan while maintaining protective immune responses against Shigella. These findings underscore the potential of the fermentor-grown STM as a safe and immunogenic vaccine platform for combating shigellosis and possibly other gastrointestinal bacterial infections. Conclusions: Further process development to optimize growth and key antigen expression as well as expanded testing in additional animal models for the assessment of protection against Shigella and Campylobacter are needed to build on these encouraging initial results. Ultimately, clinical trials are essential to evaluate the efficacy and safety of STM-based vaccines in humans. Full article

The emergence of coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has become a global issue since 2019. The prominent characteristic of SARS-CoV-2 is the presence of the spike (S) protein protruding from the virus particle envelope. The S protein is a major drug and vaccine target because it initiates the key step in infection. Medicinal herbs are a potential treatment option to enhance immunity to fight viral infections. Caesalpinia sappan L. has been reported to display promising anti-viral activities. Specifically, brazilin (BRA), a major bioactive compound in C. sappan, was reported to play a role in inhibiting viral infection. Thus, the ability of BRA as a COVID-19 treatment was tested. The S protein was used as the BRA target of this work. Understanding the binding mechanism of BRA to the S protein is crucial for future utilisation of C. sappan as a COVID-19 treatment or other coronavirus-caused pandemics. Here, we performed molecular docking of BRA onto the S protein receptor binding domain (RBD) and multimerisation (MM) pockets. Molecular dynamics (MD) simulations were conducted to study the stability of binding to glycosylated and non-glycosylated S protein constructs. BRA can bind to the Receptor-binding motif (RBM) on an RBD surface stably; however, it is too large to fit into the MM pocket, resulting in dissociation. Nonetheless, BRA is bound by residues near the S1/S2 interface. We found that glycosylation has no effect on BRA binding, as the proposed binding site is far from any glycans. Our results thus indicate that C. sappan may act as a promising preventive and therapeutic alternative for COVID-19 treatment. Full article

This review summarizes recent findings on the diverse roles of bacterial sialidases in microbial biology. Bacterial sialidases, also known as neuraminidases, are exog α-lycosidases that cleave terminal sialic acid residues from a number of complex compounds designated as sialoglycoconjugates (glycoproteins, glycolipids and oligosaccharides). Metabolically, they are involved in sialic acid catabolism, providing energy, carbon and nitrogen sources. Catabolic degradation of sialic acids is a physiological feature that can be considered an important virulence factor in pathogenic microorganisms. Sialidases play a pivotal role in host–pathogen interactions and promotion of bacterial colonization. The activity of these enzymes enables bacterial adhesion, biofilm formation, tissue invasion, and also provides immune evasion by exposing cryptic receptors and modifying immune components. Many different perspectives are being developed for the potential application of sialidases. In the field of medicine, they are being explored as appropriate targets for antimicrobials, vaccines, diagnostic preparations and in tumor immunotherapy. In the field of enzymatic synthesis, they are used for the regioselective production of oligosaccharide analogs, enzymatic separation of isoenzymes and as a tool for structural analysis of sialylated glycans, among other applications. Full article

Background: Extra-intestinal pathogenic Escherichia coli (ExPEC) represents a major global public health challenge due to its ability to cause diverse clinical infections, including urinary tract infections, bacteremia, neonatal meningitis, and sepsis. The growing prevalence of multidrug-resistant (MDR) ExPEC strains, which rapidly erode antibiotic efficacy, underscores vaccine development as a critical priority. Bioconjugate vaccines have emerged as a promising approach to mitigate ExPEC-associated infections. Methods and Results: In this study, we utilized protein glycan coupling technology (PGCT) based on oligosaccharyltransferase (OST) PglL to engineer a tetravalent bioconjugate vaccine targeting four predominant ExPEC serotypes (O1, O2, O6, and O25). We conducted a series of experiments to demonstrate the efficacy of the conjugate vaccine in eliciting humoral immune responses and inducing the production of specific antibodies against Escherichia coli O1, O2, O6, or O25 serotypes. Conclusions: This work establishes the first application of the O-linked PGCT system for engineering bioconjugate vaccines against ExPEC infections. Full article

Background. The pathogenic coronaviruses (CoVs) MERS-CoV and SARS-CoV-2, which are responsible for the MERS outbreak and the COVID-19 pandemic, respectively, continue to infect humans, with significant adverse outcomes. There is a continuing need to develop mucosal vaccines against these respiratory viral pathogens to prevent entry and replication at mucosal sites. The receptor-binding domain (RBD) of the CoV spike (S) protein is a critical vaccine target, and glycan masking is a unique approach for designing subunit vaccines with improved neutralizing activity. Methods. We evaluated the efficacy of mucosal immunity, broad neutralizing activity, and cross-protection afforded by a combined glycosylated mucosal subunit vaccine encoding the RBDs of the original SARS-CoV-2 strain (SARS2-WT-RBD), the Omicron-XBB.1.5 variant (SARS2-Omi-RBD), and MERS-CoV (MERS-RBD). Results. Intranasal administration of the three-RBD protein cocktail induced effective, durable IgA and systemic IgG antibodies specific for the S protein of these CoVs, thereby neutralizing infection by pseudotyped SARS-CoV-2-WT, Omicron-XBB.1.5, and MERS-CoV. The mucosal vaccine cocktail protected immunized mice from challenge with SARS-CoV-2 Omicron-XBB.1.5 and MERS-CoV, leading to a significant reduction in the viral titers in the lungs. By contrast, the individual glycosylated RBD proteins only induced such immune responses and neutralizing antibodies against either SARS-CoV-2 or MERS-CoV, protecting against subsequent challenge with either SARS-CoV-2 or MERS-CoV; they did not provide simultaneous protection against both CoVs. Conclusions. This study describes a unique strategy for designing efficacious mucosal subunit vaccines that induce durable mucosal immunity, cross-neutralizing activity, and cross-protection against SARS-CoV-2 and MERS-CoV, highlighting the potential for the design of mucosal vaccines against other pathogens. Full article

Cell-derived, nanoscale extracellular vesicles (EVs) have emerged as promising tools in diagnostic, therapeutic, and vaccine applications. Their unique properties including the capability to encapsulate diverse molecular cargo as well as the versatility in surface functionalization make them ideal candidates for safe and effective vehicles to deliver a range of biomolecules including gene editing cassettes, therapeutic proteins, glycans, and glycoconjugate vaccines. In this review, we discuss recent advances in the development of EVs derived from mammalian and bacterial cells for use in a delivery of carbohydrate-based protein therapeutics and vaccines. We highlight key innovations in EVs’ molecular design, characterization, and deployment for treating diseases including Alzheimer’s disease, infectious diseases, and cancers. We discuss challenges for their clinical translation and provide perspectives for future development of EVs within biopharmaceutical research and the clinical translation landscape. Full article

Background/objectives: Streptococcus agalactiae (or Group B Streptococcus, GBS) is a major cause of neonatal meningitis globally. There are 10 serotypes of GBS, which are distinguished by their capsular polysaccharide (CPS) structure, with serotypes Ia, Ib, II, III, IV and V responsible for up to 99% of infections. Currently, there are no licensed vaccines against GBS. The most developed candidates are glycoconjugate vaccines, which can be highly effective but are also expensive to produce by existing approaches and unaffordable for many parts of the world. Biosynthesis of recombinant glycans and glycoconjugates in tractable strains of bacteria offers a low-cost alternative approach to current chemical conjugation methods. Methods: In this study, we apply combinatorial hierarchical DNA assembly to the heterologous biosynthesis of GBS III, IV and V CPSs in E. coli. Each gene was removed from its native regulation, paired with synthetic regulatory elements and rebuilt from the bottom up to generate libraries of reconstituted pathways. These pathways were screened for glycan biosynthesis using serotype-specific antisera. Results: We identified several configurations that successfully biosynthesised the GBS CPSs. Furthermore, we exploited the conserved nature of the GBS CPS biosynthesis loci and the flexibility of modular DNA assembly by constructing hybrid pathways from a minimal pool of glycosyltransferase genes. We show that transferase genes with homologous function can be used interchangeably between pathways, obviating the need to clone a complete locus for each new CPS assembly. Conclusions: In conclusion, we report the first demonstration of heterologous GBS CPS IV and V biosynthesis in E. coli, a key milestone towards the development of low-cost recombinant multivalent GBS glycoconjugate vaccines. Full article

The signature of human serum IgG glycosylation is critical in the defense against pathogens. Alterations of IgG N-glycome were associated with COVID-19 (Coronavirus disease 2019) severity, although knowledge on the response to vaccination is limited. IgG N-glycome was analyzed in this study in post-COVID-19 and post-vaccination patients to reveal potential glycosylation-based alterations using hydrophilic interaction liquid chromatography (HILIC-UPLC) with fluorescence (FLR) and mass-spectrometric (MS) detection. IgG antibodies were purified from serum samples through protein G affinity chromatography followed by PNGase F digestion-based deglycosylation. The released glycans were fluorescently derivatized by procainamide labeling and purified via solid-phase extraction. Higher levels of sialylation and afucosylation were identified in post-COVID-19 patients, which was further expanded by vaccination, but only in those who were previously SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) infected. Full article

Background: Noroviruses, which cause epidemic acute gastroenteritis, and Plasmodium parasites, which lead to malaria, are two infectious pathogens that pose threats to public health. The protruding (P) domain of norovirus VP1 and the αTSR domain of the circumsporozoite protein (CSP) of Plasmodium sporozoite are the glycan receptor-binding domains of the two pathogens for host cell attachment, making them excellent targets for vaccine development. Modified norovirus P domains self-assemble into a 24-meric octahedral P nanoparticle (P₂₄ NP). Methods: We generated a unique P₂₄-αTSR NP by inserting the αTSR domain into a surface loop of the P domain. The P-αTSR fusion proteins were produced in the Escherichia coli expression system and the fusion protein self-assembled into the P₂₄-αTSR NP. Results: The formation of the P₂₄-αTSR NP was demonstrated through gel filtration, electron microscopy, and dynamic light scattering. A 3D structural model of the P₂₄-αTSR NP was constructed, using the known cryo-EM structure of the previously developed P₂₄ NP and P₂₄-VP8* NP as templates. Each P₂₄-αTSR NP consists of a P₂₄ NP core, with 24 surface-exposed αTSR domains that have retained their general conformations and binding function to heparan sulfate proteoglycans. The P₂₄-αTSR NP is immunogenic, eliciting strong antibody responses in mice toward both the norovirus P domain and the αTSR domain of Plasmodium CSP. Notably, sera from mice immunized with the P₂₄-αTSR NP bound strongly to Plasmodium sporozoites and blocked norovirus VLP attachment to their glycan receptors. Conclusion: These data suggest that the P₂₄-αTSR NP may serve as a combination vaccine against both norovirus and Plasmodium parasites. Full article

Background/Objective: Highly pathogenic (HP) H5Nx and low-pathogenicity (LP) H9N2 avian influenza viruses (AIVs) pose global threats to the poultry industry and public health, highlighting the critical need for a dual-protective vaccine. Methods: In this study, we generated a model PR8-derived recombinant H5N2 vaccine strain with hemagglutinin (HA) and neuraminidase (NA) genes from clade 2.3.2.1c H5N1 and Y439-like H9N2 viruses, respectively. To enhance the immunogenicity of the recombinant H5N2 vaccine strain, N-glycans of the HA2 subunit, NA, and M2e were modified. Additionally, we replaced M2e with avian M2e to enhance the antigenic homogeneity of AIVs for better protection. We also replaced PR8 PB2 with 01310 PB2, which is the PB2 gene derived from an LP H9N2 avian influenza virus, to eliminate pathogenicity in mammals. The productivity of the model vaccine strain (rvH5N2-aM2e-vPB2) in embryonated chicken eggs (ECEs), its potential risk of mammalian infection, and the immunogenicity associated with different inactivation methods (formaldehyde (F/A) vs. binary ethyleneimine (BEI)) were evaluated. Results: The rvH5N2-aM2e-vPB2 strain demonstrated high productivity in ECEs and exhibited complete inhibition of replication in mammalian cells. Furthermore, compared with using F/A inactivation, inactivation using BEI significantly enhanced the immune response, particularly against NA. This enhancement resulted in increased virus neutralization titers, supporting its efficacy for dual protection against H5Nx and H9N2 avian influenza viruses. Furthermore, we demonstrated that M2e-specific immune responses, difficult to induce with inactivated vaccines, can be effectively elicited with live vaccines, suggesting a strategy to enhance M2e immunogenicity in whole influenza virus vaccines. Conclusions: Finally, the successful development of the model rH5N2 vaccine strain is described; this strain provides dual protection, has potential applicability in regions where avian influenza is endemic, and can be used to promote the development of versatile H5N2 recombinant vaccines for effective avian influenza control. Full article

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in hundreds of millions of infections and millions of deaths globally. Although vaccination campaigns are mitigating the pandemic, emerging viral variants continue to pose challenges. The spike (S) protein of SARS-CoV-2 plays a critical role in viral entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, making both proteins essential targets for therapeutic and vaccine development. The glycosylation of these proteins influences their structure and function. This underscores the need for detailed site-specific glycoproteomic analysis. In this study, we characterized the N- or O-glycosylation profiles of the recombinant receptor-binding domain (RBD) of spike protein and ACE2 proteins expressed from Expi293F cells, as well as the S2 subunit of spike protein expressed in plant (N. benthamiana) cells. Using a high-resolution Orbitrap Eclipse Tribrid mass spectrometer equipped with the Ultimate 3000 RSLCnano and I-GPA (Integrated GlycoProteome Analyzer) developed in a previous study, 148 N- and 28 O-glycopeptides from RBD, 71 N-glycopeptides from the S2 subunit, and 139 N-glycopeptides from ACE2 were characterized. In addition, we report post-translational modifications (PTMs) of glycan, including mannose-6-phosphate (M6P) and GlcNAc-1-phosphate-6-O-mannose in N-glycan of RBD and ACE2, and O-acetylation in O-glycan of RBD, identified for the first time in these recombinant proteins. The relative abundance distribution according to glycosites and glycan types were analyzed by quantified site-specific N- and O (only from RBD)-glycopeptides from RBD, S2, and ACE2 using I-GPA. Asn331 for RBD, Asn1098 for S2, and Asn103 for ACE2 were majorly N-glycosylated, and dominant glycan-type was complex from RBD and ACE2 and high-mannose from S2. These findings will provide valuable insights into the glycosylation patterns that influence protein function and immunogenicity and offer new perspectives for the development of vaccines and antibody-based therapies against COVID-19. Full article

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children, elderly and immunocompromised patients worldwide. The RSV fusion (F) protein, which has 5–6 N-glycosylation sites depending on the strain, is a major target for vaccine development. Two to three of these sites are located in the p27 peptide, which is considered absent in virions. Prior research from our group showed that removing the N-glycan at position 116 (N116) in p27 led to higher neutralizing antibody responses and better protection against RSV. In this study, the effect of single, double and triple N-glycan deletion mutations in F p27 was evaluated. Surprisingly, all mutants exhibited similar expressions and functionality to the wild-type F protein. All F p27 glycomutants induced neutralizing antibodies and lowered lung viral loads after an RSV challenge in a mouse model. Although N-glycans in p27 influence immune responses, their exact role in RSV biology remains unclear. Possibly, these glycans, which are mostly conserved, play a role in other aspects of virus replication and biology. Full article