Estandarización de un ELISA para la cuantificación de anticuerpos IgG humanos contra células enteras de Bordetella pertussis / Standardization of an ELISA for the quantification of human IgG antibodies against whole cells of Bordetella pertussis

Bordetella pertussis es un patógeno exclusivo de humanos que causa la tos ferina, enfermedad respiratoria aguda que afecta principalmente a la población pediátrica. Existen dos tipos de vacunas comercializadas contra este patógeno: celulares y acelulares. Las vacunas celulares han sido extensamente utilizadas y siguen teniendo gran relevancia. El presente trabajo tuvo como objetivo la estandarización de un ELISA para la cuantificación de anticuerpos IgG contra células enteras de Bordetella pertussis. Para ello se determinó la concentración de recubrimiento, el rango lineal de la curva, los parámetros de precisión intra e interensayo, la especificidad, el valor de corte y el límite de detección. Se determinó como concentración de recubrimiento 0,5 UO/mL de células enteras. La curva estándar utilizando un suero de referencia internacional presentó un buen ajuste a una función polinómica en un intervalo entre las diluciones 1/100 y 1/24.300 con un coeficiente de correlación R2≥0,98. Los coeficientes de variación en los ensayos de precisión intra e interensayo estuvieron en los intervalos establecidos para cada uno (≤10 por ciento, ≤20 por ciento respectivamente). Los resultados obtenidos avalan el empleo de este ELISA cuantitativo para la evaluación de la respuesta a células enteras de Bordetella pertussis en ensayos clínicos(AU)

Bordetella pertussis is a pathogen exclusive to humans that causes pertussis, an acute respiratory disease that mainly affects the pediatric population. There are two types of vaccines commercially available against this pathogen: cellular and acellular. Cellular vaccines have been widely used and continue to be of great relevance. The aim of the present work was to standardize an ELISA for the quantification of IgG antibodies against whole cells of Bordetella pertussis. For this purpose, the coating concentration, the linear range of the curve, the intra- and inter-assay precision parameters, the specificity, the cut-off value and the detection limit were determined. The coating concentration was determined as 0.5 UO/mL of whole cells. The standard curve using an international reference serum presented a good fit to a polynomial function in a range between dilutions 1/100 and 1/24,300 with a correlation coefficient R2≥0.98. The coefficients of variation in the intra- and inter-assay precision tests were in the intervals established for each (≤10percent, ≤20percent respectively). The results obtained support the use of this quantitative ELISA for the evaluation of whole-cell response to Bordetella pertussis in clinical trials(AU)

Repeat-dose and local tolerance toxicity of SARS-CoV-2 FINLAY-FR-02 vaccine candidate in Sprague Dawley rats.

This study evaluates safety of FINLAY-FR-02, a vaccine candidate against SARS-CoV-2 based on the recombinant receptor binding domain conjugated to tetanus toxoid, in a preclinical, repeat-dose toxicity and local tolerance study. Sprague Dawley rats were randomly allocated to three experimental groups: control (receiving physiological saline solution); placebo (receiving all vaccine components except antigens) and vaccine group (receiving three doses of the vaccine candidate, 37.5 µg of RBD) administered intramuscularly in hind limbs at 24 h intervals during three days. We evaluated physiological condition, pain, food and water consumption, body temperature, dermal irritability, injection site temperature and inflammation, immunological response, blood chemistry, relative organ weight, histopathology and immunotoxicology. The product was well tolerated; no clinically relevant changes, pain, local effects or adverse systemic toxicological changes or deaths were observed. These preliminary results permitted the Cuban regulatory authorities to authorize clinical trials in humans.

Synthetic, Zwitterionic Sp1 Oligosaccharides Adopt a Helical Structure Crucial for Antibody Interaction.

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.

Safety and immunogenicity of the Cuban heptavalent pneumococcal conjugate vaccine in healthy infants. Results from a double-blind randomized control trial Phase I.

BACKGROUND: Cuba has a new pneumococcal conjugate vaccine candidate (PCV7-TT). This study evaluates the safety and immunogenicity in healthy infants using 2p+1 vaccination schedule. METHODS: A phase I, controlled, randomized and double blind clinical trial was designed. 30 unvaccinated healthy infants were included. 20 subjects were assigned to study group (PCV7-TT) and 10 to control group (Synflorix®) to receive the vaccines at 7, 8 months of age (primary series) and 11 months (booster dose). Blood samples were collected 30 days after second dose and post booster for antibodies measure analysis by ELISA and OPA. The statistics analysis included the frequency of occurrence for adverse events and the immune response. Non-parametric tests were used to compare the immune response. The clinical trial was published in the Cuban Public Register of Clinical Trials with code RPCEC00000173 available at http://registroclinico.sld.cu. RESULTS: Overall, the safety profile of PCV7-TT was similar to Synflorix®. Local reactions were predominant and systemic events were mild in severity. Swelling and redness were frequently associated with PCV7-TT mainly after the first dose (50% and 40% respectively). 15% and 10% of subject reported severe swelling after first dose with PCV7-TT and after second dose with Synflorix®. Mild fever (≥38-≤39), vomiting and sleep disturb were the systemic events reported. 100% of infants achieved pneumococcal IgG antibody concentrations ≥0.35 µg/ml after booster dose for serotypes 1, 14, 18C and 19F in each vaccine group. For serotypes 5, 6B and 23F, more than 80% infants vaccinated with Synflorix® or PCV7-TT achieved protective IgG GMC ≥ 0.35 µg/ml after booster dose. OPA proportion's responders to the seven common serotypes were 89.5% or more after the primary dose and 100% after booster dose in vaccinated with PCV7-TT. CONCLUSIONS: The Cuban PCV7-TT is safe, well tolerated and immunogenic in healthy infants.

Bioengineered polyester beads co-displaying protein and carbohydrate-based antigens induce protective immunity against bacterial infection.

The efficacy of protein and carbohydrate antigens as vaccines can be improved via particulate delivery strategies. Here, protein and carbohydrate antigens used in formulations of vaccines against Neisseria menigitidis were displayed on in vivo assembled polyester beads using a combined bioengineering and conjugation approach. An endotoxin-free mutant of Escherichia coli was engineered to produce translational fusions of antigens (Neisseria adhesin A (NadA) and factor H binding protein (fHbp) derived from serogroup B) to the polyhydroxybutyrate synthase (PhaC), in order to intracellularly assemble polyester beads displaying the respective antigens. Purified beads displaying NadA showed enhanced immunogenicity compared to soluble NadA. Both soluble and particulate NadA elicited functional antibodies with bactericidal activity associated with protective immunity. To expand the antigen repertoire and to design a more broadly protective vaccine, NadA-PhaC beads were additionally conjugated to the capsular polysaccharide from serogroup C. Co-delivery of surface displayed NadA and the capsular polysaccharide induced a strong and specific Th1/Th17 mediated immune response associated with functional bactericidal antibodies. Our findings provide the foundation for the design of multivalent antigen-coated polyester beads as suitable carriers for protein and polysaccharide antigens in order to induce protective immunity.

Design and Biological Assembly of Polyester Beads Displaying Pneumococcal Antigens as Particulate Vaccine.

Streptococcus pneumoniae can cause life-threatening infections mostly in infants, children, and elderly people. Capsular polysaccharide conjugate vaccines provide serotype-dependent protection against S. pneumoniae infections but fail to protect against new emerging serotypes. To overcome these limitations, pneumolysin (Ply), a serotype-independent and conserved protein was selected. As such subunit vaccines lack immunogenicity, we engineered Ply to be attached to self-assembled polyester beads in order to boost immunogenicity. To display Ply at the surface of these polyester beads, it was translationally fused to the N-terminus of the polyhydroxybutyrate (PHB) synthase (PhaC), which mediates PHB bead assembly inside recombinant Escherichia coli. We also chemically conjugated the capsular polysaccharide (CPS) 19F to isolated PHB beads to further assess their antigen carrier properties. CPS conjugated to soluble tetanus toxoid served as control. Balb/c mice immunized with Ply-PhaC beads and 19F-PhaC beads induced specific and higher IgG levels than the respective soluble counterparts. The induced IgG antibodies recognized Ply in whole cell lysates of six different serotypes of S. pneumoniae. Additionally, restimulated splenocytes from animals immunized with Ply-PhaC beads produced a balanced INF-γ/IL-17A profile unlike animals immunized with soluble Ply. The 19F-PhaC beads induced production of antibodies showing high opsonophagocytic titers against the homologous strain, serotype 19F, while CPS 19F only mixed with PhaC beads did not elicit any detectable immune response. This study provided insight into the design of PHB beads as a carrier of proteinaceous antigens and CPS in order to induce immune responses for the prevention of pneumococcal infections.

Prevalence of Pneumococcal Nasopharyngeal Carriage Among Children 2-18 Months of Age: Baseline Study Pre Introduction of Pneumococcal Vaccination in Cuba.

BACKGROUND: A new vaccine candidate against pneumococcus is being developed in Cuba, and it is a priority of the national health system. There is limited information on nasopharyngeal colonization burden, though it is essential for monitoring the impact of the vaccine. The study aims to estimate the prevalence of nasopharyngeal colonization in children 2-18 months of age and identify circulating serotypes, antimicrobial resistance and its association with selected risk factors. METHODS: A cross-sectional study was conducted between October and December 2013 in Cienfuegos municipality. Inclusion criteria were evaluated, and informed consent was obtained from the parents. Clinical and epidemiologic data were collected through a semistructured questionnaire. Nasopharyngeal swabs according to established protocols were taken. Data analysis included frequency distributions and comparison of proportions. The association between colonization and selected risk factors was assessed by multivariate analysis. RESULTS: A total of 984 children (87.2% living in urban areas) were included. The overall prevalence of colonization was 21.6%. The most frequent serotypes isolated were 6A (23.1%), 23F (10.8%), 6B (10.3%), 19F (8.5%) and 14 (3.3%). We found no resistance to ß-lactamases in circulating serotypes. Living with sibling younger than 5 years, previous respiratory infections, previous hospitalization and day-care attendance were determinants of nasopharyngeal carriage. CONCLUSIONS: The findings suggest that the burden of pneumococcal disease and colonization in Cuba could be significantly affected after vaccine introduction.

Dot Blot para determinar la identidad antigénica en vacunas conjugadas contra Streptococcus pneumoniae serotipo 19F / Dot Blot to determine the antigenic identity in conjugated vaccines against Streptococcus pneumoniae serotype 19F

Las autoridades regulatorias recomiendan el uso de técnicas de Resonancia Magnética Nuclear o técnicas serológicas para la determinación de la identidad de los antígenos presentes en las vacunas conjugadas. Con la aparición de las vacunas conjugadas multivalentes, se ha hecho necesario recurrir a técnicas inmunoquímicas con la utilización de anticuerpos monoclonales para aumentar la sensibilidad en la determinación de la identidad de los antígenos en dichas vacunas conjugadas. El objetivo del presente trabajo fue establecer las condiciones óptimas de trabajo que permitieran utilizar la técnica del Dot Blot para determinar la identidad de los antígenos en vacunas conjugadas de Streptococcus pneumoniae serotipo 19F. Para ello se estudiaron los tiempos de incubación, la influencia del reactivo en la solución de bloqueo; también las concentraciones óptimas del anticuerpo monoclonal y de los ingredientes farmacéuticos activos, así como los volúmenes de aplicación óptimos para estos y vacunas. Se utilizó un anticuerpo monoclonal contra el polisacárido capsular del serotipo 19F de neumococo. Las muestras empleadas en este trabajo fueron lotes de ingredientes farmacéuticos activos de conjugados de polisacárido capsular 19F y lotes de un candidato vacunal cubano conjugado heptavalente contra neumococos. Los resultados mostraron que para la determinación de la identidad antigénica fueron suficientes 10 µL de muestras de los principios activos a una concentración de 125 µg/mL e igual volumen para las vacunas heptavalentes. Quedó demostrado que una concentración de 1 µg/mL para el anticuerpo monoclonal y tiempos de incubación de 30 min a 37 °C fueron suficientes para la determinación. Estos resultados permiten concluir que quedaron establecidas las condiciones óptimas de trabajo para determinar la identidad antigénica por Dot Blot del polisacárido capsular de S. pneumoniae serotipo 19F presente en las vacunas conjugadas(AU)

Regulatory authorities recommend the use of NMR (Nuclear Magnetic Resonance) techniques or serological techniques to determine the identity of antigens on the conjugate vaccines. Due to the emergence of multivalent conjugate vaccines it has become necessary to use immunochemical techniques with the use of monoclonal antibodies (MAbs) in order to increase the sensitivity in determining the identity of such antigen in the conjugate vaccines. The aim of this study was to establish the optimal working conditions that would allow using Dot Blot technique to determine the identity of antigens in conjugate vaccines against Streptococcus pneumoniae serotype 19F. The incubation times, the possibility of using different reagents for blocking step were studied for this purpose; also the optimal concentrations of MAbs and active pharmaceutical ingredients (APIs), as well as the volumes of optimal application for APIs and vaccines. A monoclonal antibody against the capsular polysaccharide of S. pneumoniae serotype 19F (PsC 19F) was used. The samples used in this work, were samples of lots of APIs of monovalent conjugated PsC 19F and lots of Cuban heptavalent conjugate vaccine candidate against pneumococcus. The results showed that for the determination of the antigenic identity were optimal volumes of 10 µL of monovalent conjugate samples at 125 µg/mL and equal volume for heptavalent vaccines. For MAb was demonstrated that 1 µg/mL concentration of MAb against the PSC 19F and incubation times of 30 min at 37 °C were sufficient to successfully perform the determinations. In conclusion we can say that were established optimal working conditions to determine the antigenic identity, by Dot Blot, for the capsular polysaccharide of S. pneumoniae serotype 19F present in the APIs of monovalent conjugated and in the heptavalent conjugate vaccines(AU)

Dot Blot para determinar la identidad antigénica en vacunas conjugadas contra Streptococcus pneumoniae serotipo 19F / Dot Blot to determine the antigenic identity in conjugated vaccines against Streptococcus pneumoniae serotype 19F

Las autoridades regulatorias recomiendan el uso de técnicas de Resonancia Magnética Nuclear o técnicas serológicas para la determinación de la identidad de los antígenos presentes en las vacunas conjugadas. Con la aparición de las vacunas conjugadas multivalentes, se ha hecho necesario recurrir a técnicas inmunoquímicas con la utilización de anticuerpos monoclonales para aumentar la sensibilidad en la determinación de la identidad de los antígenos en dichas vacunas conjugadas. El objetivo del presente trabajo fue establecer las condiciones óptimas de trabajo que permitieran utilizar la técnica del Dot Blot para determinar la identidad de los antígenos en vacunas conjugadas de Streptococcus pneumoniae serotipo 19F. Para ello se estudiaron los tiempos de incubación, la influencia del reactivo en la solución de bloqueo; también las concentraciones óptimas del anticuerpo monoclonal y de los ingredientes farmacéuticos activos, así como los volúmenes de aplicación óptimos para estos y vacunas. Se utilizó un anticuerpo monoclonal contra el polisacárido capsular del serotipo 19F de neumococo. Las muestras empleadas en este trabajo fueron lotes de ingredientes farmacéuticos activos de conjugados de polisacárido capsular 19F y lotes de un candidato vacunal cubano conjugado heptavalente contra neumococos. Los resultados mostraron que para la determinación de la identidad antigénica fueron suficientes 10 µL de muestras de los principios activos a una concentración de 125 µg/mL e igual volumen para las vacunas heptavalentes. Quedó demostrado que una concentración de 1 µg/mL para el anticuerpo monoclonal y tiempos de incubación de 30 min a 37 °C fueron suficientes para la determinación. Estos resultados permiten concluir que quedaron establecidas las condiciones óptimas de trabajo para determinar la identidad antigénica por Dot Blot del polisacárido capsular de S. pneumoniae serotipo 19F presente en las vacunas conjugadas(AU)

Regulatory authorities recommend the use of NMR (Nuclear Magnetic Resonance) techniques or serological techniques to determine the identity of antigens on the conjugate vaccines. Due to the emergence of multivalent conjugate vaccines it has become necessary to use immunochemical techniques with the use of monoclonal antibodies (MAbs) in order to increase the sensitivity in determining the identity of such antigen in the conjugate vaccines. The aim of this study was to establish the optimal working conditions that would allow using Dot Blot technique to determine the identity of antigens in conjugate vaccines against Streptococcus pneumoniae serotype 19F. The incubation times, the possibility of using different reagents for blocking step were studied for this purpose; also the optimal concentrations of MAbs and active pharmaceutical ingredients (APIs), as well as the volumes of optimal application for APIs and vaccines. A monoclonal antibody against the capsular polysaccharide of S. pneumoniae serotype 19F (PsC 19F) was used. The samples used in this work, were samples of lots of APIs of monovalent conjugated PsC 19F and lots of Cuban heptavalent conjugate vaccine candidate against pneumococcus. The results showed that for the determination of the antigenic identity were optimal volumes of 10 µL of monovalent conjugate samples at 125 µg/mL and equal volume for heptavalent vaccines. For MAb was demonstrated that 1 µg/mL concentration of MAb against the PSC 19F and incubation times of 30 min at 37 °C were sufficient to successfully perform the determinations. In conclusion we can say that were established optimal working conditions to determine the antigenic identity, by Dot Blot, for the capsular polysaccharide of S. pneumoniae serotype 19F present in the APIs of monovalent conjugated and in the heptavalent conjugate vaccines(AU)