RESUMO
For the preparation of glycoconjugate vaccines, polysaccharide antigens can usually be chemically modified to generate reactive functional groups (e.g., the formation of aldehyde groups by periodate oxidation of adjacent diols) for covalent coupling with proteins. In a recent issue of JBC, Duke et al. showed that an alternative agent, galactose oxidase (GOase) isolated from the fungus Fusarium sp. can generate aldehyde groups in a unique chemoenzymatic approach to prepare a conjugate vaccine against Streptococcus pneumoniae. These findings introduce a new strategy for the design and development of glycoconjugate vaccines.
Assuntos
Glicoconjugados , Polissacarídeos , Streptococcus pneumoniae , Vacinas Conjugadas , Aldeídos , Glicoconjugados/química , Polissacarídeos/química , Polissacarídeos Bacterianos , Vacinas Conjugadas/químicaRESUMO
Multivalent vaccines addressing an increasing number of Streptococcus pneumoniae types (7-, 10-, 13-, 15-, 20-valent) have been licensed over the last 22 years. The use of polysaccharide-protein conjugate vaccines has been pivotal in reducing the incidence of invasive pneumococcal disease despite the emergence of non-vaccine serotypes. Notwithstanding its undoubtable success, some weaknesses have called for continuous improvement of pneumococcal vaccination. For instance, despite their inclusion in pneumococcal conjugate vaccines, there are challenges associated with some serotypes. In particular, Streptococcus pneumoniae type 3 remains a major cause of invasive pneumococcal disease in several countries.Here a deep revision of the strengths and weaknesses of the licensed pneumococcal conjugate vaccines and other vaccine candidates currently in clinical development is reported.
Assuntos
Infecções Pneumocócicas , Vacinas Pneumocócicas , Humanos , Vacinas Pneumocócicas/uso terapêutico , Streptococcus pneumoniae , Infecções Pneumocócicas/prevenção & controle , Infecções Pneumocócicas/epidemiologia , Vacinação , Vacinas Conjugadas/uso terapêutico , Anticorpos AntibacterianosRESUMO
Meningococcal meningitis remains a substantial cause of mortality and morbidity worldwide. Until recently, countries in the African meningitis belt were susceptible to devastating outbreaks, largely attributed to serogroup A Neisseria meningitidis (MenA). Vaccination with glycoconjugates of MenA capsular polysaccharide led to an almost complete elimination of MenA clinical cases. To understand the molecular basis of vaccine-induced protection, we generated a panel of oligosaccharide fragments of different lengths and tested them with polyclonal and monoclonal antibodies by inhibition enzyme-linked immunosorbent assay, surface plasmon resonance, and competitive human serum bactericidal assay, which is a surrogate for protection. The epitope was shown to optimize between three and six repeating units and to be O-acetylated. The molecular interactions between a protective monoclonal antibody and a MenA capsular polysaccharide fragment were further elucidated at the atomic level by saturation transfer difference NMR spectroscopy and X-ray crystallography. The epitope consists of a trisaccharide anchored to the antibody via the O- and N-acetyl moieties through either H-bonding or CH-π interactions. In silico docking showed that 3-O-acetylation of the upstream residue is essential for antibody binding, while O-acetate could be equally accommodated at three and four positions of the other two residues. These results shed light on the mechanism of action of current MenA vaccines and provide a foundation for the rational design of improved therapies.
Assuntos
Epitopos/imunologia , Meningite Meningocócica/prevenção & controle , Vacinas Meningocócicas/imunologia , Neisseria meningitidis/imunologia , Polissacarídeos Bacterianos/imunologia , Acetilação , Adolescente , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Criança , Ensaios Clínicos Fase II como Assunto , Cristalografia por Raios X , Feminino , Humanos , Imunogenicidade da Vacina , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Masculino , Meningite Meningocócica/imunologia , Meningite Meningocócica/microbiologia , Vacinas Meningocócicas/uso terapêutico , Simulação de Acoplamento Molecular , Estudos Multicêntricos como Assunto , Polissacarídeos Bacterianos/química , Ensaios Clínicos Controlados Aleatórios como Assunto , Sorogrupo , Ensaios de Anticorpos Bactericidas Séricos , Vacinas Conjugadas/imunologia , Vacinas Conjugadas/uso terapêuticoRESUMO
The increasing diffusion of antimicrobial resistance (AMR) across more and more bacterial species emphasizes the urgency of identifying innovative treatment strategies to counter its diffusion. Pathogen infection prevention is among the most effective strategies to prevent the spread of both disease and AMR. Since their discovery, vaccines have been the strongest prophylactic weapon against infectious diseases, with a multitude of different antigen types and formulative strategies developed over more than a century to protect populations from different pathogens. In this review, we review the main characteristics of vaccine formulations in use and under development against AMR pathogens, focusing on the importance of administering multiple antigens where possible, and the challenges associated with their development and production. The most relevant antigen classes and adjuvant systems are described, highlighting their mechanisms of action and presenting examples of their use in clinical trials against AMR. We also present an overview of the analytical and formulative strategies for multivalent vaccines, in which we discuss the complexities associated with mixing multiple components in a single formulation. This review emphasizes the importance of combining existing knowledge with advanced technologies within a Quality by Design development framework to efficiently develop vaccines against AMR pathogens.
RESUMO
Several glycoconjugate vaccines have been licensed or are currently in clinical development to prevent bacterial infections. Here we report the development of a single analytical assay to quantify the conjugated saccharide content, as alternative to two separated total and free (unconjugated) saccharide assays used so far, for a quadrivalent conjugate vaccine containing meningococcal serogroup A polysaccharide (α-1,6-linked N-acetylmannosamine phosphate repeating unit partly O-acetylated at position C3 or C4) coupled with CRM197 protein. The results confirm a high linear correlation among the two approaches (conjugated saccharide content vs. difference of total saccharide and free saccharide). Conjugated saccharide content estimation is therefore demonstrated to be a suitable method to monitor the product quality of vaccines containing meningococcal serogroup A conjugate antigen, in the final filled presentation as demonstrated here and potentially on the bulk conjugate before formulation.
Assuntos
Infecções Meningocócicas , Vacinas Meningocócicas , Neisseria meningitidis , Anticorpos Antibacterianos , Glicoconjugados , Humanos , Infecções Meningocócicas/prevenção & controle , Potência de Vacina , Vacinas ConjugadasRESUMO
Glycoconjugate vaccines are among the most effective interventions for preventing several serious infectious diseases. Covalent linkage of the bacterial capsular polysaccharide to a carrier protein provides CD4+ T cells with epitopes that facilitate a memory response to the polysaccharide. Classically, the mechanism responsible for antigen processing was thought to be similar to what was known for hapten-carrier conjugates: protease digestion of the carrier protein in the endosome and presentation of a resulting peptide to the T cell receptor on classical peptide-recognizing CD4+ T cells. Recently, an alternative mechanism has been shown to be responsible for the memory response to some glycoconjugates. Processing of both the protein and the polysaccharide creates glycopeptides in the endosome of antigen-presenting cells. For presentation, the peptide portion of the glycopeptide is bound to MHCII, allowing the covalently linked glycan to activate carbohydrate-specific helper CD4+ T cells (Tcarbs). Herein, we assessed whether this same mechanism applies to conjugates prepared from other capsular polysaccharides. All of the glycoconjugates tested induced Tcarb-dependent responses except that made with group C Neisseria meningitidis; in the latter case, only peptides generated from the carrier protein were critical for helper T cell recognition. Digestion of this acid-sensitive polysaccharide, a linear homopolymer of α(2 â 9)-linked sialic acid, to the size of the monomeric unit resulted in a dominant CD4+ T cell response to peptides in the context of MHCII. Our results show that different mechanisms of presentation, based on the structure of the carbohydrate, are operative in response to different glycoconjugate vaccines.
Assuntos
Imunidade Adaptativa/imunologia , Glicoconjugados/imunologia , Polissacarídeos Bacterianos/imunologia , Vacinas Conjugadas/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Animais , Formação de Anticorpos/imunologia , Antígenos/imunologia , Epitopos/imunologia , Feminino , Citometria de Fluxo , Glicoconjugados/farmacologia , Vacinas Meningocócicas/imunologia , Vacinas Meningocócicas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Ácido N-Acetilneuramínico , Neisseria meningitidis/imunologia , Polissacarídeos Bacterianos/farmacologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas Conjugadas/farmacologiaRESUMO
Vaccination is the most cost-effective way to control disease caused by encapsulated bacteria; the capsular polysaccharide (CPS) is the primary virulence factor and vaccine target. Neisseria meningitidis (Nm) serogroups B, C, Y and W all contain sialic acid, a common surface feature of human pathogens. Two protein-based vaccines against serogroup B infection are available for human use while four tetravalent conjugate vaccines including serogroups C, W and Y have been licensed. The tetravalent Menveo® conjugate vaccine is well-defined: a simple monomeric structure of oligosaccharides terminally conjugated to amino groups of the carrier protein CRM197. However, not only is there a surprisingly low limit for antigen chain attachment to CRM197, but different serogroup saccharides have consistently different CRM197 loading, the reasons for which are unclear. Understanding this phenomenon is important for the long-term goal of controlling conjugation to prepare conjugate vaccines of optimal immunogenicity. Here we use molecular modeling to explore whether antigen flexibility can explain the varying antigen loading of the conjugates. Because flexibility is difficult to separate from other structural factors, we focus on sialic-acid containing CPS present in current glycoconjugate vaccines: serogroups NmC, NmW and NmY. Our simulations reveal a correlation between Nm antigen flexibility (NmW > NmC > NmY) and the number of chains attached to CRM197, suggesting that increased flexibility enables accommodation of additional chains on the protein surface. Further, in silico models of the glycoconjugates confirm the relatively large hydrodynamic size of the saccharide chains and indicate steric constraints to further conjugation.
Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Vacinas Meningocócicas/química , Simulação de Dinâmica Molecular , Ácido N-Acetilneuramínico/química , Humanos , Vacinas Meningocócicas/imunologiaRESUMO
Neisseria meningitidis is a major cause of bacterial meningitidis worldwide. Children less than five years and adolescents are particularly affected. Nearly all invasive strains are surrounded by a polysaccharide capsule, based on which, 12 N. meningitidis serogroups are differentiated. Six of them, A, B, C, W, X, and Y, cause the vast majority of infections in humans. Mono- and multi-valent carbohydrate-based vaccines against meningococcal infections have been licensed or are currently in clinical development. In this mini-review, an overview of the past and present approaches for producing meningococcal glycoconjugate vaccines is provided.
Assuntos
Glicoconjugados/química , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/imunologia , Humanos , Infecções Meningocócicas/microbiologia , Neisseria meningitidis/classificação , Neisseria meningitidis/imunologia , Vacinas Conjugadas/imunologiaRESUMO
The capsular polysaccharide of the human pathogen Group B Streptococcus is a key virulence factor and vaccine candidate that induces protective antibodies when conjugated to carrier proteins. It consists of long polymeric chains of oligosaccharide repeating units, and each of the ten capsular serotypes described so far presents a unique chemical structure with distinct antigenic properties; therefore, broad protection against this pathogen could be achieved by a combination of ten glycoconjugates. Capsular polysaccharide biosynthesis and assembly follow a polymerase-dependent pathway that is widespread in encapsulated bacteria and is encoded by a polycistronic operon. Here we exploited the sequence similarity between the capsule operons of types V and IX to generate hybrid polysaccharides incorporating epitopes of both serotypes in a single molecule, by co-expressing their specific CpsM, O, I glycosyltransferases in a single isolate. Physicochemical and immunochemical methods confirmed that an engineered strain produced a high molecular weight chimeric polysaccharide, combining antigenic specificities of both type V and IX. By optimizing the copy number of key glycosyltransferase genes, we were able to modulate the ratio between type-specific epitopes. Finally, vaccination with chimeric glycoconjugates significantly decreased the incidence of disease in pups born from immunized mice challenged with either serotype. This study provides proof of concept for a new generation of glycoconjugate vaccines that combine the antigenic specificity of different polysaccharide variants in a single molecule, eliciting a protective immune response against multiple serotype variants.
Assuntos
Cápsulas Bacterianas/imunologia , Polissacarídeos Bacterianos/imunologia , Vacinas Estreptocócicas/imunologia , Streptococcus agalactiae/imunologia , Vacinas Combinadas/imunologia , Animais , Anticorpos Monoclonais , Proteínas de Bactérias/imunologia , Feminino , Engenharia Genética , Glicoconjugados , Humanos , Imunidade Materno-Adquirida , CamundongosRESUMO
Recent structural studies demonstrated that the epitope recognized by a monoclonal antibody representative of the protective response against the type III group B Streptococcus polysaccharide was comprised within 2 of the repeating units that constitute the full-length native structure. In the current study, we took advantage of this discovery to design a novel vaccine based on multivalent presentation of the identified minimal epitope on a carrier protein. We show that highly glycosylated short oligosaccharide conjugates elicit functional immune responses comparable to those of the full-length native polysaccharide. The obtained results pave the way to the design of well-defined glycoconjugate vaccines based on short synthetic oligosaccharides.
Assuntos
Epitopos/química , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Infecções Estreptocócicas/prevenção & controle , Vacinas Estreptocócicas/imunologia , Streptococcus agalactiae , Animais , Configuração de Carboidratos , Epitopos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Oligossacarídeos/imunologiaRESUMO
Invited for the cover of this issue is the group of Roberto Adamo at GlaxoSmithKline Research Center, Siena, and colleagues at The University of the Basque Country and Basque Research Technology Alliance. The image depicts a tactical plan with the different elements of the research as part of the team. Read the full text of the article at 10.1002/chem.202000284.
Assuntos
Polissacarídeos/síntese química , Streptococcus/química , Humanos , Polissacarídeos/química , Vacinas SintéticasRESUMO
Identification of glycan functional epitopes is of paramount importance for rational design of glycoconjugate vaccines. We recently mapped the structural epitope of the capsular polysaccharide from typeâ III Groupâ B Streptococcus (GBSIII), a major cause of invasive disease in newborns, by using a dimer fragment (composed of two pentasaccharide repeating units) obtained by depolymerization complexed with a protective mAb. Although reported data had suggested a highly complex epitope contained in a helical structure composed of more than four repeating units, we showed that such dimer conjugated to a carrier protein with a proper glycosylation degree elicited functional antibodies comparably to the full-length conjugated polysaccharide. Here, starting from the X-ray crystallographic structure of the polysaccharide fragment-mAb complex, we synthesized a hexasaccharide comprising exclusively the relevant positions involved in binding. Combining competitive surface plasmon resonance and saturation transfer difference NMR spectroscopy as well as in-silico modeling, we demonstrated that this synthetic glycan was recognized by the mAb similarly to the dimer. The hexasaccharide conjugated to CRM197 , a mutant of diphtheria toxin, elicited a robust functional immune response that was not inferior to the polysaccharide conjugate, indicating that it may suffice as a vaccine antigen. This is the first evidence of an X-ray crystallography-guided design of a synthetic carbohydrate-based conjugate vaccine.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Epitopos/química , Glicoconjugados/química , Polissacarídeos/imunologia , Streptococcus agalactiae/química , Streptococcus agalactiae/imunologia , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologia , Humanos , Vacinas SintéticasRESUMO
Despite substantial progress in the prevention of group B Streptococcus (GBS) disease with the introduction of intrapartum antibiotic prophylaxis, this pathogen remains a leading cause of neonatal infection. Capsular polysaccharide conjugate vaccines have been tested in phase I/II clinical studies, showing promise for further development. Mapping of epitopes recognized by protective antibodies is crucial for understanding the mechanism of action of vaccines and for enabling antigen design. In this study, we report the structure of the epitope recognized by a monoclonal antibody with opsonophagocytic activity and representative of the protective response against type III GBS polysaccharide. The structure and the atomic-level interactions were determined by saturation transfer difference (STD)-NMR and X-ray crystallography using oligosaccharides obtained by synthetic and depolymerization procedures. The GBS PSIII epitope is made by six sugars. Four of them derive from two adjacent repeating units of the PSIII backbone and two of them from the branched galactose-sialic acid disaccharide contained in this sequence. The sialic acid residue establishes direct binding interactions with the functional antibody. The crystal structure provides insight into the molecular basis of antibody-carbohydrate interactions and confirms that the conformational epitope is not required for antigen recognition. Understanding the structural basis of immune recognition of capsular polysaccharide epitopes can aid in the design of novel glycoconjugate vaccines.
Assuntos
Cápsulas Bacterianas/química , Epitopos/química , Oligossacarídeos/química , Polissacarídeos Bacterianos/química , Streptococcus agalactiae/química , Animais , Configuração de Carboidratos , Cristalografia por Raios X , Camundongos , CoelhosRESUMO
This review focuses upon the use of nitroso Diels-Alder reactions as a structural complexity generating reaction that has been so far a quite scarcely treated topic, despite its potential. In particular, the use of N-acyl-1,2-dihydropyridines as a non-symmetrical diene component in nitroso Diels-Alder reactions encompasses an initial diversification of pathways giving rise to different cycloadducts (direct and inverse). Selective elaborations of these cycloadducts, basically using a reagent-based approach, deliver a discrete number of structurally diverse compounds, including some original heterobicyclic scaffolds and functionalized heterocycles. This forward synthetic planning allowed the individuation of a new biologically active compound based on a novel oxadiaza-bicyclic-[3.3.1]-nonene scaffold which is still under preclinical evaluation.
Assuntos
Reação de Cicloadição , Di-Hidropiridinas/química , Compostos Nitrosos/química , Ciclização , Estrutura MolecularRESUMO
The direct catalytic α-amidoalkylation of dihydroquinolines with aldehydes bearing oxygen functionalities at different positions in a Mannich-type reaction has been studied. ß-Alkoxy-aldehyde 1d gave high enantioselectivity, albeit with an inherently poor diastereoselectivity, while the use of α-alkoxy aldehydes 1c was detrimental also to enantioselectivity. Mannich-type reactions have been studied for the first time using new chiral carbohydrate-derived aldehydes 1a,b showing a reactivity markedly influenced by the presence of water. The chiral glycidic backbone showed a slight but significant influence on the overall stereochemical outcome only when present in α-position of the aldehyde. The absolute stereochemistry of the products was studied by electronic circular dichroism (ECD) spectra and compared with theoretical calculations. ECD analysis easily provides the absolute configuration of 1,2-dihydroquinoline derivatives such as quinoline-1(2H)-carboxylates.
RESUMO
Glycoconjugate vaccines obtained by chemical linkage of a carbohydrate antigen to a protein are part of routine vaccinations in many countries. Licensed antimicrobial glycan-protein conjugate vaccines are obtained by random conjugation of native or sized polysaccharides to lysine, aspartic or glutamic amino acid residues that are generally abundantly exposed on the protein surface. In the last few years, the structural approaches for the definition of the polysaccharide portion (epitope) responsible for the immunological activity has shown potential to aid a deeper understanding of the mode of action of glycoconjugates and to lead to the rational design of more efficacious and safer vaccines. The combination of technologies to obtain more defined carbohydrate antigens of higher purity and novel approaches for protein modification has a fundamental role. In particular, methods for site selective glycoconjugation like chemical or enzymatic modification of specific amino acid residues, incorporation of unnatural amino acids and glycoengineering, are rapidly evolving. Here we discuss the state of the art of protein engineering with carbohydrates to obtain glycococonjugates vaccines and future perspectives.
Assuntos
Proteínas de Bactérias/química , Vacinas Bacterianas/química , Glicoconjugados/química , Vacinas Conjugadas/química , Animais , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/genética , Vacinas Bacterianas/síntese química , Vacinas Bacterianas/genética , Técnicas de Química Sintética/métodos , Glicoconjugados/síntese química , Glicoconjugados/genética , Humanos , Engenharia de Proteínas/métodos , Vacinas Conjugadas/genéticaRESUMO
The particular nature of tetrahydropyrido[4,3- e]-1,4,2-dioxazines of type 1 allows the regio- and stereoselective obtainment of substituted N-carbamoyl tetrahydropyridines by common reducing agents. A completely novel, biologically active, bicyclic 1,3-diaza-4-oxa-[3.3.1]-nonene scaffold can be generated by the use of lithium triethylborohydride through unprecedented cascade syn-SN2' reduction/carbamate reduction/cyclization reactions. The remarkable regioselectivity switches in the allylic reduction process have been rationalized with the aid of computational studies.
RESUMO
The incidence of infectious diseases caused by several bacterial pathogens such as Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis, has been dramatically reduced over the last 25 years through the use of glycoconjugate vaccines. The structures of the bacterial capsular polysaccharide (CPS) antigens, extracted and purified from microbial cultures and obtained with very high purity, show that many of them are decorated by O-acetyl groups. While these groups are often considered important for the structural identity of the polysaccharides, they play a major role in the functional immune response to some vaccines such as meningococcal serogroup A and Salmonella typhi Vi, but do not seem to be important for many others, such as meningococcal serogroups C, W, Y, and type III Group B Streptococcus. This review discusses the O-acetylation status of CPSs and its role in the immunological responses of these antigens.
Assuntos
Vacinas Bacterianas/imunologia , Polissacarídeos Bacterianos/imunologia , Acetilação , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/metabolismo , Glicoconjugados/imunologia , Humanos , Neisseria meningitidis Sorogrupo C/imunologia , Salmonella typhi/imunologia , Staphylococcus aureus/imunologia , Streptococcus pneumoniae/imunologiaRESUMO
Bioconjugates represent an emerging class of medicines, which offer therapeutic opportunities overtaking those of the individual components. Many novel bioconjugates have been explored in order to address various emerging medical needs. The last decade has witnessed the exponential growth of new site-selective bioconjugation techniques, however very few methods have made the way into human clinical trials. Here we discuss various applications of site-selective conjugation in biomedicines, including half-life extension, antibody-drug conjugates, conjugate vaccines, bispecific antibodies and cell therapy. The review is intended to highlight both the progress and challenges, and identify a potential roadmap to address the gap.
Assuntos
Imunoconjugados , Sistemas de Liberação de Medicamentos , HumanosRESUMO
Carbohydrate-based vaccines are among the safest and most effective vaccines and represent potent tools for prevention of life-threatening bacterial infectious diseases, like meningitis and pneumonia. The chemical conjugation of a weak antigen to protein as a source of T-cell epitopes generates a glycoconjugate vaccine that results more immunogenic. Several methods have been used so far to characterize the resulting polysaccharide-protein conjugates. However, a reduced number of methodologies has been proposed for measuring the degree of saccharide conjugation at the possible protein sites. Here we show that detailed information on large proteins conjugated with large polysaccharides can be achieved by a combination of solution and solid-state NMR spectroscopy. As a test case, a large protein assembly, l-asparaginase II, has been conjugated with Neisseria meningitidis serogroup C capsular polysaccharide and the pattern and degree of conjugation were determined.