The genetic structure of Neisseria meningitidis populations in Cuba before and after the introduction of a serogroup BC vaccine.

We investigated the population genetics in collections of meningococci sampled in Cuba during the period 1983-2005, thereby covering a period before and after the introduction of an antimeningococcal B-C vaccine. A total of 163 case isolates and 210 isolates from healthy carriers were characterized by multilocus sequence typing (MLST) and sequence determination of porA, porB and fetA genes. A total of 56 sequence types (STs) including 28 new STs were identified among these isolates. The analysis of surface antigens revealed variants 3-1 and 3-8 to be prevalent for porB; variant F5-1 was the most common FetA epitope, and variants 19 and 15 corresponded to the prevalent variable regions 1 (VR1) and VR2 PorA epitopes, respectively. The strongest associations between specific surface protein variants and clonal complexes were detected in lineages ST-32 and ST-53. All ST-32 complex isolates possessed porB3 alleles, and the most frequent antigen combination among ST-32 complex isolates was P1.19,15;F5-1. Variants PorB3-64 at PorB and P1.30 at PorA VR2, in combination with the PorA VR1 variants P1.12-1, P1.7 and P1.7-2 as well as the FetA variants F1-2 and F1-7, dominated the ST-53 complex organisms. Furthermore, we observed a statistically significant association between the most frequent porA, porB and fetA alleles and strain invasiveness. Finally, this study showed that the application of VA-MENGOC-BC((R)), the Cuban antimeningococcal vaccine, reduced the number and frequency of the hypervirulent Clonal Complexes ST-32 and ST-41/44, and also impacted on other lineages. The vaccine also affected the genetic composition of the carrier-associated meningococcal isolates. The number of carrier isolates belonging to hypervirulent lineages decreased significantly after vaccination, and ST-53, a sequence type common in carriers, became the predominant ST.

Clonal distribution of disease-associated and healthy carrier isolates of Neisseria meningitidis between 1983 and 2005 in Cuba.

In response to epidemic levels of serogroup B meningococcal disease in Cuba during the 1980s, the VA-MENGOC-BC vaccine was developed and introduced into the National Infant Immunization Program in 1991. Since then the incidence of meningococcal disease in Cuba has returned to the low levels recorded before the epidemic. A total of 420 Neisseria meningitidis strains collected between 1983 and 2005 in Cuba were analyzed by multilocus sequence typing (MLST). The set of strains comprised 167 isolated from disease cases and 253 obtained from healthy carriers. By MLST analysis, 63 sequence types (STs) were identified, and 32 of these were reported to be a new ST. The Cuban isolates were associated with 12 clonal complexes; and the most common were ST-32 (246 isolates), ST-53 (86 isolates), and ST-41/44 (36 isolates). This study also showed that the application of VA-MENGOC-BC, the Cuban serogroup B and C vaccine, reduced the frequency and diversity of hypervirulent clonal complexes ST-32 (vaccine serogroup B type-strain) and ST-41/44 and also affected other lineages. Lineages ST-8 and ST-11 were no longer found during the postvaccination period. The vaccine also affected the genetic composition of the carrier-associated meningococcal isolates. The number of carrier isolates belonging to hypervirulent lineages decreased significantly after vaccination, and ST-53, a sequence type common in carriers, became the predominant ST.

Proteomic study via a non-gel based approach of meningococcal outer membrane vesicle vaccine obtained from strain CU385: a road map for discovering new antigens.

This work presents the results from a study of the protein composition of outer membrane vesicles from VA-MENGOC-BC (Finlay Institute, Cuba), an available vaccine against serogroup B Neisseria meningitidis. Proteins were identified by means of SCAPE, a 2DE-free method for proteome studies. More than one hundred proteins were detected by tandem liquid chromatographymass spectrometry analysis of fractions enriched in peptides devoid of histidine or arginine residues, providing a detailed description of the vaccine. A bioinformatic analysis of the identified components resulted in the identification of 31 outer membrane proteins and three conserved hypothetical proteins, allowing the cloning, expression, purification and immunological study of two of them (NMB0088 and NMB1796) as new antigens.

Comparison of phenotypically indistinguishable but geographically distinct Neisseria meningitidis Group B isolates in a serum bactericidal antibody assay.

The "gold standard" assay for measuring serologic protection against Neisseria meningitidis group B (MenB) is the serum bactericidal antibody (SBA) assay. Of vital importance to the outcome of the SBA assay is the choice of the target strain(s), which is often chosen on the basis of phenotype or genotype. We therefore investigated the effect on the results produced by the SBA assay of using phenotypically indistinguishable but geographically distinct MenB isolates. Nine PorA P1.19,15 and 11 PorA P1.7-2,4 MenB isolates were incorporated into the SBA assay using human complement and were assayed against sera obtained either before or after outer membrane vesicle vaccination. Large differences in the results produced by the isolates in the SBA assay were demonstrated. These included differences as great as 5.8-fold in SBA geometric mean titers and in the proportions of subjects with SBA titers of >/=4. Ranges of as many as 9 SBA titers were achieved by individual sera across the panels of isolates. To determine the reasons for the differences observed, investigations into the expression of capsular polysaccharide, PorA, PorB, Opc, and lipooligosaccharide (LOS) and into LOS sialylation were completed. However, minor differences were found between strains, indicating similar expression and no antigen masking. These results have implications for the choice of MenB target strains for inclusion in future studies of MenB vaccines and highlight the requirement for standardization of target strains between laboratories.

New meningococcal C polysaccharide-tetanus toxoid conjugate Physico-chemical and immunological characterization.

The polysaccharides (Ps) are thymus-independent 2 (TI-2) antigens and poor immunogens in infants and young children; as a result of this delayed response to Ps antigens during ontogeny, infants and young children are highly susceptible to infections caused by encapsulated bacteria. Meningococcal group C polysaccharide (PsC)-proteins conjugate vaccines have been reported to induce significant serum IgG antibodies and immunologic memory in infants resulting in very effective vaccines. We describe here the obtainment, by a new method, of a neoglycoconjugate intended to immunize against Neisseria meningitidis serogroup C, its characterization by physico-chemical methods, including (1)H NMR and fluorescence spectroscopy methods, as well as the characterization of the immune response induced in mice by such conjugate. Amine groups generated by basic hydrolysis in the PsC were successfully conjugated to carboxyl groups of tetanus toxoid (TT), using carbodiimide-mediated coupling. The specific anti-Ps IgG and anti-Ps IgG subclasses (IgG1 and IgG2a) were measured by ELISA methods, the bactericidal activity in sera and the cytokines response (IFNgamma or IL5) in spleen cell of mice immunized with conjugated and native Ps were evaluated. The (1)H NMR spectra and the result obtained by the fluorescence spectroscopy method showed that the PsC and TT maintained structural identity after conjugation process. Conjugated PsC elicited an increase of anti-PsC IgG responses, anti-PsC IgG subclass (IgG1, IgG2a), an eight-fold increase in bactericidal activity in sera of mice immunized with conjugate compared with native PsC, was also observed. Higher titres of IFNgamma were observed in mice immunized with conjugated Ps. These results indicated that, the PsC and TT maintained its chemical and antigenic structure after the conjugation process. A change in the immunological pattern of responses of PsC, from TI-2 to a thymus-dependent (TD) pattern, was also demonstrated.

Cuban Meningococcal BC Vaccine: Experiences & Contributions from 20 Years of Application.

This paper reviews 20 years of experience and scientific contributions of the Cuban meningococcal BC vaccine (VA-MENGOC-BC®) obtained by the Finlay Institute in Havana, Cuba. The vaccine is the first of its type in the world that is safe, effective, and commercially available for preventing meningococcal disease caused by serogroup B meningococcus; it is also effective against serogroup C. VA-MENGOC-BC® has shown satisfactory results, with no serious adverse events, after application of approximately 55 million doses in some 15 countries. Also included is background information on meningococcal disease, as well as the main characteristics of VA-MENGOC-BC®, the strategy used for controlling meningococcal disease and its prevention in Cuba, and a summary of the main scientific results obtained in basic research, development, clinical evaluation, and post-marketing results (safety, efficacy-effectiveness, post-vaccination adverse events, etc.) in Cuba and elsewhere.

Immunogenicity and safety of three doses of a bivalent (B:4:p1.19,15 and B:4:p1.7-2,4) meningococcal outer membrane vesicle vaccine in healthy adolescents.

An experimental bivalent meningococcal outer membrane vesicle (OMV) vaccine (B:4:P1.19,15 and B:4:P1.7-2,4) has been developed to provide wide vaccine coverage particularly of the circulating strains in Europe. A randomized, controlled phase II study (study identification number, 710158/002; ClinicalTrials.gov identifier number, NCT00137917) to evaluate the immunogenicity and safety of three doses of the OMV vaccine when given to healthy 12- to 18-year-olds on a 0-2-4 month (n = 162) or 0-1-6 month schedule (n = 159). A control group received two doses of hepatitis A and one of conjugated meningococcal serogroup C vaccine on a 0-1-6 month schedule (n = 157). Immune response, defined as a fourfold increase in serum bactericidal titer using a range of vaccine-homologous or PorA-related and heterologous strains, was determined for samples taken before and 1 month after vaccination; assays were performed at two laboratories. As measured at the GlaxoSmithKline (GSK) laboratory, the OMV vaccine induced an immune response against homologous or PorA-related strains (in at least 51% of subjects against strains of serosubtype P1.19,15 and at least 66% against strains of serosubtype P1.7-2,4) and against a set of three heterologous strains (in 28% to 46% of subjects). Both laboratories showed consistent results for immune response rates. The OMV vaccine had a similar reactogenicity profile for each schedule. Pain preventing normal activities occurred in approximately one-fifth of the subjects; this was significantly higher than in the control group. The immune responses induced by the bivalent OMV vaccine demonstrated the induction of bactericidal antibodies against the vaccine-homologous/PorA-related strains but also against heterologous strains, indicating the presence of protective antigens in OMVs and confirming the potential of clinical cross-protection.

Mucosal immune responses to meningococcal C polysaccharide-protein conjugate in mice.

Mucosal delivery of vaccines represents an attractive approach because this is a region of first contact point for inhaled antigens. We have obtained a meningococcal group C polysaccharide-tetanus toxoid conjugate (MGCP-TT) and evaluated it for intranasal route in mice. The conjugate was obtained by a developed method in our laboratory. The specific IgA in saliva and specific IgA and IgG in serum were measured by ELISA methods and bactericidal antibodies in sera against a meningococcal group C strain were measured. The conjugated elicited a significant increase in anti-MGCP salivary IgA and serum IgG and bactericidal antibodies concentrations, while specific serum IgA was not observed. These results indicated that after conjugation, there was a change in the responses for MGCP from thymus-independent to thymus-dependent and that it was effective by intranasal route.

Outer membrane vesicles of the VA-MENGOC-BC vaccine against serogroup B of Neisseria meningitidis: Analysis of protein components by two-dimensional gel electrophoresis and mass spectrometry.

Neisseria meningitidis is a Gram-negative bacterium responsible for significant mortality worldwide. While effective polysaccharides-based vaccines exist against serogroups A, C, W135, and Y, no similar vaccine is suitable for children under 4 years against disease caused by serogroup B strains. Therefore, major vaccine efforts against this serogroup are based on outer membrane vesicles (OMVs), containing major outer membrane proteins. The OMV-based vaccine produced by the Finlay Institute in Cuba (VA-MENGOC-BC) contributed to the rapid decline of the epidemic in this Caribbean island. While the content of major proteins in this vaccine has been discussed, no detailed work of an outer membrane proteomic map of this, or any other, commercially available OMV-derived product has been published so far. Since OMVs exhibit a large bias toward a few major proteins and usually contain a high content of lipids, establishing the adequate conditions for high resolution, 2-DE of this kind of preparation was definitely a technical challenge. In this work, 2-DE and MS have been used to generate a proteomic map of this product, detailing the presence of 31 different proteins, and it allows the identification of new putative protective protein components it contains.

Inmunogenicidad inducida por la vacuna antimeningocócica VA-MENGOC-BC® contra la cepa de N. meningitidis ATCC C11 en adolescentes después de 12 años de vacunados / Immunogenecity induced by the VA-MENGOC-BC® antimeningococcal vaccine against the ATCC C11 N. meningitidis strain in adolescents 12 years after being vaccinated

Se estudió la respuesta de anticuerpos inducida por la vacuna antimeningocócica cubana VA-MENGOC-BC® contra la cepa ATCC C11 mediante Ensayo Bactericida del Suero y ELISA, a 184 adolescentes de un Politécnico de Ciego de Ávila, que habían sido inmunizados en campañas masivas 12 años antes. Se realizaron extracciones de sangre antes de aplicar la primera dosis (T0), 4 semanas después de esta (T1) y 4 semanas después de la segunda dosis (T2). Después de 12 años de la vacunación, 25 por ciento de los adolescentes presentó títulos bactericidas ≥ 1:8 frente a la cepa evaluada. Por ELISA, 78 por ciento mostró una concentración de anticuerpos superior al límite de detección contra el polisacárido capsular del meningococo C. Los porcentajes de seroconversión posterior a la primera dosis fueron 59 por Ensayo Bactericida del Suero y 82 por ELISA. No hubo diferencias significativas (p> 0,05) entre los resultados obtenidos después de la primera y segunda dosis por ambos ensayos. La reinmunización con 2 dosis de la vacuna no provocó hiporrespuesta frente a la cepa ATCC C11 en este grupo de edad

[Immunogenecity induced by the VA-MENGOC-BC antimeningococcal vaccine against the ATCC C11. N. meningitidis strain in adolescents 12 years after being vaccinated]. / Inmunogenicidad inducida por la vacuna antimeningocócica VA-MENGOC-BC contra la cepa de N. meningitidis ATCC C11 en adolescentes después de 12 años de vacunados.

The antibodies' response induced by the VA-MENGOC-BC Cuban antimeningococcal vaccine against the ATCC C11 strain was studied by Bactericidal Serum Trial and ELISA among 184 adolescents from a polytechnic, in Ciego de Avila, that had been immnunized in mass campaings 12 years before. Blood samples were taken before administering the first dose (T0), 4 weeks later (T1), and 4 weeks after the second dose (T2). 12 years after vaccination, 25% of the adolescents presented bactericidal titers > or =1:8 against the evaluated strain. 78% showed a concentration of antibodies over the limit of detection against the meningococcus C capsular polyssacharide. The percentages of seroconversion after the first dose were 59 by Bactericidal Serum Trial and 82 by ELISA. There were no significant differences (p > 0.05) between the results obtained after the first and second dose by boths trials. The reimmnunization with 2 doses of the vaccine did not cause hyporesponse against the ATCC C11 strain in this age group.

Portadores de Neisseria meningitidis en niños de una escuela primaria

Se realizó, con la autorización de la Dirección Municipal de Educación, Dirección Municipal de Salud y el consentimiento informado de los padres, un estudio transversal descriptivo en 318 niños de la Escuela "Mártires del Corynthia"; con el propósito de conocer la prevalencia de portadores de meningococo en niños de edad escolar, determinar los marcadores epidemiológicos de las cepas aisladas y establecer la posible relación existente entre el portador y las variables como edad, sexo, antecedente de infección respiratoria aguda, hacinamiento, amigdalectomía, efecto inhibitorio de la flora acompañante y el estado secretor de antígenos ABH en la saliva. A todos, se les tomó exudado nasofaríngeo y una muestra de saliva. Además, los padres llenaron una encuesta donde se indagó sobre los factores de riesgo a investigar. Se detectó 6,9 % de portadores de meningococo y predominaron las cepas NA:NT:P1.NST:L3,7,9. Los factores de riesgo que dieron resultados estadísticamente significativos respecto a la condición de portador de Neisseria meningitidis fueron: edad, antecedente de infección respiratoria aguda y la presencia de Streptococcus pneumoniae y Neisseria lactamica de la flora bacteriana acompañante en la nasofaringe de los niños investigados.

A cross-sectional and descriptive study was conducted among 318 children from the "Mártires del Corynthia" Primary School under the authorization of the Municipal Division of Education and the informed consent of their parents aimed at knowing the prevalence of meningoccoco carriers in school children, determining the epidemiological markers of the isolated strains and establishing the possible relation existing between the carrier and variables, such as age, sex, acute respiratory infection history, hacinamiento, amigdalectomy, inhibitory effect of of the accompanying flora and the secretory state of ABH antigens in saliva. All of them underwent nasopharyngeal exudate and a saliva sample was taken. In adition, the paents were surveyed about the risks factors to be investigated. 6.9 % of meningoccoco carriers were found and the NA:NT:P1:NST:L3,7,9 strains predominated. The risk factors with statistically significant results regarding the condition of carrier Neisseria meningitidis carrier were age, acute respiratory infection history, and the presence of Streptococcus pneumoniae and Neisseria lactamica of the accompanying bacterial flora in the nasopharynx of the children under study.

[Carriers of Neisseria meningitidis among children from a primary school]. / Portadores de Neisseria meningitidis en niños de una escuela primaria.

A cross-sectional and descriptive study was conducted among 318 children from the "Mártires del Corynthia" Primary School under the authorization of the Municipal Division of Education and the informed consent of their parents aimed at knowing the prevalence of meningoccoco carriers in school children, determining the epidemiological markers of the isolated strains and establishing the possible relation existing between the carrier and variables, such as age, sex, acute respiratory infection history, hacinamiento, amigdalectomy, inhibitory effect of of the accompanying flora and the secretory state of ABH antigens in saliva. All of them underwent nasopharyngeal exudate and a saliva sample was taken. In adition, the paents were surveyed about the risks factors to be investigated. 6.9 % of meningoccoco carriers were found and the NA:NT:P1:NST:L3,7,9 strains predominated. The risk factors with statistically significant results regarding the condition of carrier Neisseria meningitidis carrier were age, acute respiratory infection history, and the presence of Streptococcus pneumoniae and Neisseria lactamica of the accompanying bacterial flora in the nasopharynx of the children under study.

Obtención de gammaglobulina hiperinmune antimeningocócica y su uso potencial en el tratamiento de la enfermedad meningocócica en Cuba: resultados preliminares / Attainment of antimeningococcal hyperimmune gammaglobulin and its potential use in the treatment of menigococcal disease in Cuba: preliminary results

A partir de plasmas de donantes inmunizados con la vacuna antimeningocócica de producción nacional, se obtuvo gammaglobulina hiperinmune, mediante un método de fraccionamiento alcohólico. El producto final obtenido tuvo altos títulos de anticuerpos específicos contra determinantes antigénicos de la Neisseria menigitidis, así como una alta actividad bactericida, lo cual unido a que cunplía los requerimientos de la OMS para gammaglobulinas de uso endovenoso, permitió el inicio de su utilización en el tratamiento de pacientes con enfermedad meningocócica. El objetivo del presente trabajo se circunscribe a presentar, de manera preliminar, los resultados concernientes a la obtención del preparado, a su caracterización inicial y potencialidad terapéutica. En un próximo trabajo se presentarán in extenso comparaciones con otros métodos de obtención, así como la casuística clínica y una mejor fundamentación de las ideas de mecanismos. Un amplio colectivo de médicos intensivistas, así como investigadores, labora ya en otra segunda fase