Quantitative NMR for the structural analysis of novel bivalent glycoconjugates as vaccine candidates.

Novel unimolecular bivalent glycoconjugates were assembled combining several functionalized capsular polysaccharides of Streptococcus pneumoniae and Neisseria meningitidis to a carrier protein by using an effective strategy based on the Ugi 4-component reaction. The development of multivalent glycoconjugates opens new opportunities in the field of vaccine design, but their high structural complexity involves new analytical challenges. Nuclear Magnetic Resonance has found wide applications in the characterization and impurity profiling of carbohydrate-based vaccines. Eight bivalent conjugates were studied by quantitative NMR analyzing the structural identity, the content of each capsular polysaccharide, the ratios between polysaccharides, the polysaccharide to protein ratios and undesirable contaminants. The qNMR technique involves experiments with several modified parameters for obtaining spectra with quantifiable signals. In addition, the achieved NMR results were combined with the results of colorimetric assay and Size Exclusion HPLC for assessing the protein content and free protein percentage, respectively. The application of quantitative NMR showed to be efficient to clear up the new structural complexities while allowing the quantitative assessment of the components.

NMR line-fitting quantification of polysaccharide N-acylurea-based modification in glycoconjugates of Salmonella Typhi Vi polysaccharide.

The polysaccharides modification via carbodiimide reaction is one of the most applied methods for obtaining conjugated vaccines against Salmonella enterica. However, N-acylurea carbodiimide adduct generated in the process is a critical impurity in carbohydrate-based vaccines. A quantitative NMR method was developed for assessing the N-acylurea carbodiimide adduct impurity. The procedure was based on line-fitting facilities for processing the NMR signals on complex spectra. The method showed good linearity, accuracy and precision under inter-operator variation (relative standard deviation <5%). Copyright © 2017 John Wiley & Sons, Ltd.

Safety and Immunogenicity of Cuban Antipneumococcal Conjugate Vaccine PCV7-TT in Healthy Adults.

INTRODUCTION Pneumococcal infections are a major cause of morbidity and mortality and are associated with considerable economic burden on health systems. To prevent pneumococcal infections, 7-valent conjugate vaccines have been available for over a decade; more recently, 10- and 13-valent conjugate vaccines have been formulated, which are more immunogenic than vaccines with capsular polysaccharides only. In Cuba, a new vaccine candidate has been developed, PCV7-TT, a conjugate of tetanus toxoid with antigens of seven of the serotypes of Streptococcus pneumoniae with highest circulation in Cuba and in the world: 1, 5, 6B, 14, 18C, 19F and 23F. OBJECTIVE Assess the safety of the vaccine candidate PCV7-TT in healthy adults and conduct a preliminary assessment of its immunogenicity. METHODS A phase I, double-blind clinical trial was performed at the National Toxicology Center in Havana, Cuba. Healthy male volunteers aged 18-35 years were randomly assigned to two groups: 20 received the vaccine candidate PCV7-TT and 20 the polyvalent antipneumococcal vaccine PNEUMO-23 used as control, each in a single intramuscular dose. To assess safety, the occurrence of adverse events was monitored for 30 days following inoculation. To explore immunogenicity, concentrations of serotype-specific antibodies was quantified before and 30 days after inoculation, as well titers of opsonophagocytic antibodies. (National Clinical Trial Registry RPCEC00000133) RESULTS Local adverse events were pain, redness, induration, increased sensitivity to touch, and warmth in the injection area. Pain was registered in 70% of individuals who received PCV7-TT and in 75% of those vaccinated with PNEUMO-23. Reported systemic adverse events were general malaise, headache and drowsiness. All adverse events appeared in the first 72 hours post inoculation and lasted no longer than 3 days. One event was reported that was classified as severe in intensity and serious in consequences, but it was unrelated to vaccination appendicitis in one individual inoculated with the control vaccine. Before vaccination, all participants but one had antibody concentrations =0.20 µg/ml against the vaccine strains; after vaccination 100% of individuals were positive and the concentrations of antibodies increased in previously positive volunteers. Some individuals had opsonophagocytic antibodies against serotypes 1, 14, 19F and 23F before vaccination, with highest concentrations against serotypes 14 and 19F. After vaccination, the percent of individuals with opsonophagocytic titers >1:8 for all serotypes in the vaccine was >50% in both groups. CONCLUSIONS A single dose of candidate vaccine PCV7-TT was safe when used in healthy adults. Preliminary results showed that it was able to activate an immune response against the serotypes of Streptococcus pneumoniae used. KEYWORDS Invasive pneumococcal diseases, pneumococcal vaccines, conjugate vaccines, immunization, randomized clinical trial, safety, Cuba.

Resonancia magnética nuclear: nuevas aplicaciones en la cuantificación y la evaluación de intermediarios de vacunas basadas en polisacáridos / Nuclear Magnetic Resonance: new applications in the quantification and assessment of polysaccharide-based vaccine intermediates

La resonancia magnética nuclear (RMN) constituye una fuerte alternativa para estudios estructurales, evaluación de identidad y cuantificación de ingredientes farmacéuticos activos (IFA). En las dos últimas décadas la aplicación de la resonancia magnética nuclear cuantitativa (RMNc) tuvo un creciente impacto en la cuantificación de compuestos, fundamentalmente orgánicos. Varios resultados obtenidos mediante la RMNc han ido creando un lugar para la técnica en la industria biofarmacéutica. La RMNc incluye experimentos de RMN con algunos parámetros modificados, con el objetivo de obtener señales cuantificables. Basada en algunos de los reportes más relevantes, la presente revisión aborda algunas de las aplicaciones de la RMN para vacunas basadas en polisacáridos o glicoproteínas. El trabajo hace especial énfasis en detallar algunos aspectos que caracterizan la RMNc, así como sus aplicaciones(AU)

Nuclear Magnetic Resonance has become the choice for structural studies, identity assays and simultaneous quantification of active pharmaceutical ingredient of different polysaccharide-based vaccine. In the last two decades, the application of quantitative Nuclear Magnetic Resonance had an increasing impact to support several quantification necessities. The technique involves experiments with several modified parameters in order to obtain spectra with quantifiable signals. The present review is supported by some recent relevant reports and it discusses several applications of NMR in carbohydrate-based vaccines. Moreover, it emphasizes and describes several parameters and applications of quantitative Nuclear Magnetic Resonance(AU)

Caracterización de conjugados inmunogénicos de polisacárido capsular Streptococcus pneumoniae serotipo 14 / Characterization of capsular polysaccharide immunogenic conjugates from Streptococcus pneumoniae serotype 14

Las vacunas conjugadas que consisten en polisacáridos bacterianos unidos a través de un enlace covalente a una proteína portadora, han tenido un gran impacto en los esquemas de vacunación infantil, disminuyendo de forma dramática la incidencia de infecciones bacterianas. En el caso de Streptococcus pneumoniae, a pesar de que se han descrito más de 90 serotipos basados en la estructura de las cápsulas polisacarídicas y que al menos 23 tienen una importancia clínica demostrada, solo un número limitado de siete, o más recientemente 10 y 13, están incluidos en las vacunas conjugadas licenciadas. Por otra parte, la necesidad creciente de estas vacunas en el mundo requiere la incorporación de nuevos productores que se enfrentan a una elevada complejidad tecnológica, pues en todo el procedimiento de conjugación no se pueden afectar las características estructurales por las que el polisacárido es reconocido inmunológicamente. Este trabajo implementó un procedimiento de conjugación para el polisacárido de la cápsula de Streptococcus pneumoniae serotipo 14. El procedimiento comprendió la fragmentación, oxidación peryódica y posterior conjugación del polisacárido a anatoxina tetánica o diftérica. Cada intermedio fue caracterizado por métodos físico-químicos. En todas las reacciones se obtuvieron rendimientos superiores al 50%. Los conjugados generaron altos títulos de anticuerpos específicos de tipo IgG y memoria inmunológica. Se concluyó que el procedimiento permitió la obtención de conjugados inmunogénicos de serotipo 14(AU)

Conjugate vaccines consisting of bacterial polysaccharides linked through a covalent bond to a carrier protein have a major impact on childhood immunization schemes which have dramatically decrease the incidence of bacterial infections. In the case of Streptococcus pneumoniae more than 90 serotypes have been reported, based on the structure of the polysaccharide capsules and at least 23 of them have demonstrated clinical importance. A limited number of 7 or more recently 10 and 13 are included in licensed conjugate vaccines. On the other hand, the increasing need for these vaccines worldwide requires the incorporation of new manufacturers who are facing highly complex technology since the entire conjugation process can not affect the structural features for which the polysaccharide is immunologically recognized. Our paper provides a conjugation procedure for the capsular polysaccharide of Streptococcus pneumoniae serotype 14. The process includes fragmentation, peryodic oxidation and subsequent conjugation to tetanus toxoid or diphtheria toxoid to the polysaccharide, each intermediate was characterized by physico-chemical methods. Yields higher than 50% were obtained in all reactions. The conjugates generated high titers of IgG specific antibodies and immunological memory. In conclusion, the procedure allows immunogenic conjugates of serotype 14(AU)

Quantitative proton nuclear magnetic resonance evaluation and total assignment of the capsular polysaccharide Neisseria meningitidis serogroup X.

Neisseria meningitidis constitutes the main cause of meningococcal disease in infants. Serogroups A, B, C, W135, Y, and X have the higher incidence in young children and teenagers. The use of polyvalent conjugate carbohydrate-based vaccines has decreased the meningococcal infection around the world. Recently, the serogroup X has been found to be responsible of different outbreaks of meningococcal diseases, mainly in "Meningitis Belt" of Africa and the structure of the repetitive unit of the capsular polysaccharide has been confirmed through a monodimensional (13)C NMR study. No further characterization studies have been carried out, especially with the use of other nuclei. In this paper a novel method for quantification of the N. meningitidis serogroup X by proton qNMR is reported. Deep characterization of the serogroup X polysaccharide was also carried out by combination of correlation experiments involving (13)C, (1)H, and (31)P nuclei.

Direct validation of NGcGM3 ganglioside as a new target for cancer immunotherapy.

OBJECTIVE: The target concept means not only an aberrant expression of a particular molecule in tumour tissues but also evidence of a clear therapeutic advantage, as a consequence of immune-intervention, in an antigen-positive relevant tumour model. Since we reported the presence of NGcGM3 ganglioside in human breast tumours years ago and though Phase I clinical trials of a ganglioside containing vaccine have been conducted, a definitive direct validation of this peculiar molecule as target for cancer immunotherapy has remained unperformed. METHODS: Two animal models were used: leghorn chickens and C57BL/6 mice. The murine 3LL-D122 cell line, the derived subcutaneous tumours and metastatic lung lesions were processed for gangliosides identification. Active immunotherapy experiments in the 3LL-D122 spontaneous lung metastasis model were performed with NGcGM3/VSSP vaccine prepared by conjugation of NGcGM3 with the outer membrane proteins of Neisseria meningitides. RESULTS: The 3LL-D122 Lewis lung carcinoma results were consistent with an increased expression of NGcGM3 from primary tumours to metastatic lesions, as observed in human breast cancer samples. Both vaccines, prepared with synthetic or natural-source-derived ganglioside, showed similar anti-tumour and immunogenicity profiles. Finally, a clear involvement of NK1.1(+) cells and CD8(+) T cells in the anti-metastatic effect elicited by the vaccine was manifested. CONCLUSIONS: While 'proof of concept' Phase II and III clinical trials with the NGcGM3/VSSP vaccine in cancer patients are currently ongoing these results reasonably sustain the validation of this peculiar ganglioside as a novel target for cancer immunotherapy.

UltramicroELISA para la deteccion de anticuerpos IgM anti M.leprae / Ultramicro-Elisa for the detection of IgM antibodies anti M. leprae

La disponibilidad del sistema Ultramicroanalitico (SUMA) y de un antigeno especie-especifico del M. leprae obtenido mediante sintesis quimica, permitio la normalizacion y validacion de un ultramicroELISA para la deteccion de anticuerpos IgM especificos a esta micobacteria. El analisis de 433 sueros de banco de sangue y 265 sueros usados para validar el metodo y clasificados en un grupo control de donantes de banco de sangre (100), un grupo de pacientes tuberculosos (50), un grupo de enfermos de lepra (65) y un grupo de contactos de estos enfermos (50), mostro la especificidad del ensayo para evidenciar la infeccion con el M. leprae. Los resultados obtenidos del estudio adicional de 140 muestras de suero de contactos de enfermos estuvieron estrechamente correlacionados (r = 0,98) con los resultados obtenidos por la tecnica de microELISA convencional. La utilizacion del SUMA no solo permite un notable ahorro de reactivos si no ademas facilita la lectura, calculo, validacion y almacenamiento automatico de los resultados.