Estandarización en Colombia de una prueba ELISA para la evaluación de los niveles séricos de anticuerpos IgG contra diez serotipos de Streptococcus pneumoniae / An immunoenzymatic test for IgG antibody levels against 10 serotypes of Streptococcus pneumoniae

Introducción. Streptococcus pneumoniae es causante de gran morbimortalidad en niños pequeños y ancianos. Sin embargo, en Colombia no está disponible una prueba que evalúe la respuesta humoral a la vacunación específica contra este microorganismo Objetivo. Estandarizar en Colombia un ensayo inmunoenzimático para evaluar los niveles séricos de anticuerpos IgG contra diez serotipos de S. pneumoniae en respuesta a la vacunación específica y caracterizar esta respuesta en individuos sanos de nuestra población. Materiales y métodos. Se hizo un ELISA en fase sólida utilizando como antígenos los polisacáridos capsulares 1, 3, 4, 5, 6B, 9V, 14, 18, 19F y 23F de S. pneumoniae. Resultados. Los sueros de referencia y control reaccionaron fuertemente contra los polisacáridos evaluados, especialmente contra 14 y 19F. En los cinco niños sanos evaluados, los polisacáridos 5 y 19F presentaron los mayores títulos antes de la vacunación. Antes de la vacunación en los niños, y antes y después de la vacunación en los adultos, los polisacáridos 14 y 19F reaccionaron fuertemente. Para todos los polisacáridos, excepto para el 5, existe una relación inversa entre títulos altos de anticuerpos IgG antes de la vacunación y la razón de incremento de los títulos después de la misma. Conclusión. Esta prueba ELISA cuantifica de forma confiable los niveles de IgG sérica contra diez serotipos de S. pneumoniae y, de acuerdo con los resultados obtenidos en individuos sanos de nuestra población, en este trabajo se validan los parámetros internacionales para considerar adecuada la respuesta a la vacuna 23-valente contra este microorganismo.

Introduction. Streptococcus pneumoniae is a major cause of morbi-mortality in early childhood and elderly. However, a test to measure the antibody responses after specific vaccination is not available in Colombia. Objective. An immunoenzymatic test was standardized for the measurement of serum IgG levels against 10 serotypes of S. pneumoniae in response to the specific vaccination. Material and methods. Capsular polysaccharides 1, 3, 4, 5, 6B, 9V, 14, 18, 19F, 23F of S. pneumoniae were used as antigens in a solid-phase ELISA. These responses were characterized in a randomized selected healthy individuals from a Colombian population. Results. The reference and control sera showed great reactivity against all the polysaccharides evaluated, especially against polysaccharide 14 and 19F. The lowest reactivity in these two sera was observed against polysaccharide 3 and 4. Among the children evaluated, polysaccharide 5/19F showed the highes pre-vaccination reactivity, and polysaccharide 14/19F showed the highest post-vaccination reactivity. Among the adults, polysaccharides 14 and 19F showed the greatest reactivity pre- and post-vaccination. For all the polysaccharides (excepting polysaccharide 5), an inverse association among high polysaccharide-specific pre-vaccination- and the increase of post-vaccination-IgG levels was observed. Conclusion. This ELISA test reliably quantifies the serum levels of specific IgG against 10 serotypes of S. pneumoniae. According to the responses by healthy individuals, the current study validates parameters used internationally as an adequate the response to the 23-valent pneumococcal vaccine.

Vacinação contra Haemophilus influenzae tipo b: proteção a longo prazo / Haemophilus influenzae type b vaccination: long term protection

OBJETIVO: Identificar as evidências sobre o impacto da vacina conjugada para Haemophilus influenzae tipo b (Hib) na epidemiologia da doença invasiva por Hib. FONTE DOS DADOS: Pesquisa nas bases de dados do MEDLINE, LILACS, publicações técnicas de organizações internacionais, diretrizes nacionais e internacionais, nos últimos 15 anos (1991-2005), utilizando os seguintes unitermos: Haemophilus influenzae type b, immunization, impact, effectiveness. Foram incluídas as publicações que apresentaram informação para atender o objetivo deste artigo. Artigos publicados em período anterior ao da pesquisa e citados em referências dos artigos incluídos foram analisados quanto à apresentação de informação de interesse. SíNTESE DOS DADOS: A introdução da vacina conjugada para Hib produziu grande declínio na incidência de casos de doença invasiva por Hib nos diversos países em que seu uso foi incorporado à rotina de vacinação das crianças. No entanto, o ressurgimento de casos com doença invasiva por Hib tem mobilizado vários investigadores na busca das possíveis explicações para esses eventos, bem como a identificação das medidas a serem implementadas para evitar o reaparecimento da doença. CONCLUSÕES: O uso da vacina conjugada para Hib em escala populacional tem sido extremamente efetivo. No entanto, mudanças no esquema vacinal poderão ser necessárias para a manutenção do controle da doença invasiva por Hib, frente ao atual cenário epidemiológico das infecções pelo Hib.

Response to polysaccharide antigens in patients with ataxia-telangiectasia

OBJECTIVE: To analyze the production of antibodies to polysaccharide antigens in patients with ataxia-telangiectasia.PATIENTS AND METHODS: We used the ELISA technique to measure the levels of IgG antibodies to serotypes 1, 3, 5, 6B, 9V and 14 of Streptococcus pneumoniae in 14 patients with ataxia-telangiectasia before and after immunization with 23-valent polysaccharide vaccine. Adequate response to individual polysaccharide can be defined as a postimmunization antibody titer equal to or greater than 1.3 µg/ml or as a minimum fourfold increase over the baseline (preimmunization) value. RESULTS: Six (43%) patients showed an absent response to all serotypes analyzed. Four patients showed adequate response to only one serotype, one patient to two serotypes, two patients to three serotypes and only one patient to four out of six serotypes analyzed. No patient had adequate response to all serotypes tested. Postimmunization pneumococcus IgG levels were higher than preimmunization levels to all serotypes analyzed, except for serotype 3. In spite of this, the mean postimmunization levels were lower than 1.3 µg/ml in all serotypes, except for serotype 14. Mean increment was less than four in all serotypes analyzed. CONCLUSION: Our results suggest that patients with ataxia-telangiectasia are at a high risk of having an impaired response to pneumococcus, which may be one of the causes of recurrent sinopulmonary infections in these patients.

Vaccines based on the cell surface carbohydrates of pathogenic bacteria

Vacinas glicoconjugadas, cujo carboidrato da superfície de um microrganismo está covalentemente ligado a uma proteína carreadora, vêm sendo consideradas como efetivas para gerar respostas imunes que previnem um grande número de doenças. A tecnologia é genérica e aplicável a vários patógenos, se os anticorpos contra os carboidratos de superfície forem capazes de proteger contra a infecção. Três vacinas contra Haemophilus influenzae tipo b, Neissseria meningitidis Grupo C e sete sorotipos de Streptococcus pneumoniae já foram licenciadas e muitas outras estão em desenvolvimento. Este artigo discute o racional para o desenvolvimento e uso de vacinas glicoconjugadas; os mecanismos pelos quais elas induzem respostas imune dependentes de célula T e suas implicações para o seu desenvolvimento; o papel dos métodos físico-químicos na caracterização e no controle de qualidade dessas vacinas; e os produtos novos que estão em desenvolvimento.

Conjugate vaccines--a breakthrough in vaccine development.

The encapsulated bacteria Streptococcus pneumoniae (the pneumococcus), Neisseria meningitidis (the meningococcus) and Haemophilus influenzae type b (Hib) are the main causes of purulent meningitis, the peak incidence of which is seen in the first two years of life. The polysaccharide capsule of these bacteria is an essential virulence determinant, and antibodies to it are protective, suggesting that a polysaccharide vaccine could prevent these diseases. The young child is, however, unable to respond with antibody production to these polysaccharides, making such vaccines useless in infancy. Conjugation of the polysaccharide to a protein carrier has proven a way to solve the problem. Immunization of infants with such a Hib conjugate vaccine was shown in 1987 to result in the desired antibody production and protection from Hib meningitis and bacteremia. The Hib vaccine is now a part of national infant immunization programs in large parts of Europe, the Americas and Australia, and has resulted in the virtual disappearance of Hib disease from these areas. A group C meningococcal and 7-valent pneumococcal vaccine, available since 2000, are likewise proving highly effective in preventing bacteremic disease. Further advantages of the conjugate vaccines are their ability to elicit immunologic memory and to reduce asymptomatic carriage of the bacteria, resulting in marked herd immunity. This paper was delivered as a lecture in January 2003 in Bangkok on the occasion of the Prince Mahidol Award for a life's work in the field of vaccinology.

Efficacy of Purified Vi Polysaccharide Typhoid Vaccine

This experiment was conducted to assess the efficacy of typhoid vaccine newly produced by purifying Vi antigen of Salmonella typhi. With Karber method, LD50 of challenging organism (S. typhi ty2) was determined as 6.31 CFU/mouse, and then the organism was used for the study. With Probits method, ED50 of the vaccine was determined as 0.016 microgram / 0.5 ml / mouse. The ELISA titer (0.5097+/-0.0606) was 4 times in the group treated with high dose (0.25 microgram/0.5ml) as in control (0.1113+/-0.0110). Six major protein bands of 66, 55, 35, 33, 18, and 9 kd were detected in Western blot analysis with serum of a vaccine treated mouse, whereas only one weak band of about 35 kd was detected with serum of a control mouse. We concluded that typhoid vaccine produced by purifying Vi antigen of S. typhi very effectively prevent S. typhi infection in mice.

Effectiveness of a mass immunization campaign against serogroup C meningococci in children in the federal state of Santa Catarina, Brazil

In addition to vaccine efficacy studies, there is a pressing need to evaluate vaccine effectiveness in a way that takes into account the limitations of health care systems in certain settings. An attempt to reach this objective was exemplified by a vaccination campaign against serogroup C meningococci in the federal state of Santa Catarina, in Brazil. A polysaccharide vaccine against serogroup C meningococci was administered to all individuals between 6 months and 14 years of age in March, 1996, in the municipalities that had the highest incidence of meningococcal disease in the previous year. All cases of the disease due to this serogroup observed in Santa Catarina during a 1-year period before and after the vaccination were included in the analysis. The cumulative incidence rate ratio was calculated for the unvaccinated compared to the vaccinated area. As a second step, the ratio of this quantity for the period before and after the vaccination, i.e. the ratio of the rate ratios (RRR), was calculated. One minus RRR was used to estimate the vaccine effectiveness. In the general population, the vaccine effectiveness was 74.3 (95 confidence intervals 52.7 to 99.6). In children 6 months to 14 years, vaccine effectiveness was 93.1 (85.2 to 100). Vaccine effectiveness could not be confirmed within more specific age bands, probably due to the lack of statistical power. It is concluded that group C meningococcal vaccine is effective in reducing the occurrence of meningococcal disease in children 6 months to 14 years of age, and that the ratio of rate ratios (RRR) in a useful method to evaluate vaccine effectiveness.

Molecular characterization and optimization of VI-capsular polysaccharide of salmonella typhi TY6S production in bioreactor

The role of Vi-capsular polysaccharide [Vi-CPS] in human immunity against infection caused by Salmonella typhi is well known. The downstream process of purification generally causes depolymerization of Vi-CPS to a nonimmunogenic low molecular weight form. In the present study, a standard strain of Sal. typhiTy6s was grown under submerge cultural conditions in a pilot-plant scale of 90 liter fermentor. At the late exponential growth phase, crude polysaccharide was obtained from fermentation broth by detergent-phenol extraction method and purified by ultracentrifuge differentiation technique. Analytical data reveals that by optimization of fermentation parameters, not only was the yield of production increased from 1.6 mg/L to 4.9 mg/L, but also the polysaccharide retained its native molecular stability and immunogenicity. Therefore, purified Vi-CPS can be regarded as a reliable immunogen to control typhoid fever in man

Dosagem de níveis de anticorpos contra polissacarídeos capsulares de Streptococcus pneumoniae após vacinaçäo de pacientes HIV positivos com vacina pneumocócica 23-valente / Quantification of antibody levels anti-Streptococcus pneumoniae capsular polysaccharide in HIV-infected patients after vaccination with the 23-valent vaccine

Streptococcus pneumoniae é o agente causal mais comum de pneumonias bacterianas e bacteremias nao micobacterianas em indivíduos infectados pelo HIV, estimando-se que esses pacientes sejam de 100 a 300 vezes mais susceptíveis à doença pneumocóccica invasiva, se comparados a indivíduos nao infectados pelo HIV. Desde 1989 o Centers for Disease Control and Prevention, EUA indica a vacina pneumocócia 23-valente na populaçao HIV+. No entanto, vários estudos encontraram uma resposta diminuída à vacinaçao nesta populaçao, principalmente nos indivíduos imunossuprimidos. Em contrapartida, outros estudos observaram respostas semelhantes aos controles normais, sendo sua indicaçao ainda polêmica. Realizamos a dosagem de anticorpos contra polissacarídeos capsulares de Streptococcus pneumoniae sorotipos 1, 3, 5, 6B, 9V e 14 antes e após imunizaçao com vacina pneumocócica 23-valente de 15 indivíduos voluntários normais HIV-e de 42 indivíduos HIV+, distribuídos em três grupos pela contagem de LTCD4+ (Grupo I > 500 células/mm3, Grupo II entre 200 e 499 células/mm3 e Grupo III < 200 células/mm3). Observamos que os títulos de anticorpos contra os polissacarídeos capsulares do Streptococcus pneumoniae, antes da vacinaçao, sao semelhante entre os indivíduos normais e os portadores do vírus HIV, independentemente do grau de imunodepressao. Verificamos também que a resposta à imunizaçao com a vacina pneumocócica 23-valente, aos antígenos capsulares dos sorotipos testados, é semelhante entre os controles normais e os indivíduos HIV+, mesmo nos pacientes com LTCD4+ < 200 céls/mm3, independentemente do uso de anti-retrovirais. Nenhum dos grupos respondeu de forma adequada, ao sorotipo 3; o sorotipo que induziu a melhor resposta em todos os grupos foi o 14.

C substance--specific latex agglutination for early & rapid detection of Streptococcus pneumoniae in blood cultures.

A slide agglutination test was developed using latex particles coated with antiserum against the C substance, a common antigen for all serotypes of Streptococcus pneumoniae. This test was used for the rapid identification of pneumococci in blood culture broths which contained Gram positive cocci (GPC) in pairs or short chains on smear examination. Of 238 consecutive blood cultures with GPC tested, 72 were positive for Strep. pneumoniae by the latex test and conventional methods. The remaining 166 cultures were negative for both these, indicating a 100 per cent specificity and sensitivity for the test.

Antibody response after immunization of Brazilian children with serogroup C meningococcal polysaccharide noncovalently complexed with outer membrane proteins

We have studied the antibody response of Brazilian vaccines to C meningococcal polysaccharide (C-PS) after one or two doses of a vaccine composed of C-PS, outer membrane proteins of B meningococci and aluminum hydroxide. Total IgG, IgG1 and IgG2 as well as bactericidal activity mediated by complement were measured in serum samples from children 3 to 83 months of age (post-vaccination IgG, IgG1 and IgG2 levels of 2.4 to 13.4 µg/ml; less than 18 to 67.8 U/ml and less than 8 to 106.8U/ml, respectively) and from individuals 10 to 14 years of age (post-vaccination IgG, IgG1 and IgG2 levels of 14.6 µg/ml, 23,7 U/ml and 112.0 U/ml, respectively). The antibody response, measured as IgG levels, was age-dependent. Although high antibody levels were demonstrableby enzyme-linked immunosorbent assay (ELISA), bactericidal activity was not demonstrable (less than 1:4) in serum from children aged less than 24 months. A significant bactericidal activity was detected in serum of children older than 49 months of age and in individuals 10 to 14 years of age. A predominance of IgG2 was observed in post-vaccination serum samples from children belonging to those two age groups. The antibody concentration sufficient to confer protection as well as the possible causes of the poor correlation observed between ELISA and bactericidal activity results are discussed

Immunogenicity of Vi capsular polysaccharide vaccine evaluated for three years in Korea

The immunogenicity of a single dose of Salmonella typhi(S.typhi) Vi capsular polysaccharide(CPS) vaccine was evaluated before, and at 1, 3, 12, and 36 months after vaccination. Eighty-five adults(20-28 years of age) and sixty-four children(8-16 years of age) received a single dose of 25 micrograms Vi CPS vaccine intramuscularly, and antibody titers to Vi CPS were measured by passive hemagglutination. Of 149 vaccinees, 138(92.6%) showed seroconversion at 1 month after vaccination, and then 138 out of 141(97.9%) did at 3 months. Of 137 vaccinees, 116(84.7%) maintained a persistent rise in Vi antibody titer 12 months after vaccination, and 55 out of 100(55.0%) had a 4-fold or greater rise at 36 months. No significant adverse reactions were observed. Booster injection may be needed 3-5 years after vaccination.

Reaction of the sera of the Korean children free from Hib invasive diseases against H. influenzae type B capsular polysaccharide antigen

The purpose of our experiment is to examine the level of anti-Haemophilus influenza polysaccharide antibody titer in the Korean population. Using ELISA, the level of Hib-PS antibodies in 384 infants and children who were all free from Hib invasive diseases, was tested. And the blood of 50 mothers within 24 hours of delivery and cord blood from their respective full-term neonates was also tested. The transport of Hib-PS IgG and IgG subclasses in paired sera from mothers and neonates was also measured. The titer of Hib-PS IgG varies with age. At birth the mean optical density of cord blood was 1.028; however, it declined to 0.609 up to 6 months and further decline was noted up to 2 years to 0.488. Then the mean O.D. remained around 0.5 from 3 to 14 years of age. The mean O.D. of Hib-PS IgG in the mothers blood was 0.856. The ratio of mean O.D. of anti-Hib PS IgG antibody in the cord blood to that in the maternal blood was 1.20. The mean optical densities of IgG subclasses were: 1.18 for anti-Hib PS IgG1, 1.07 for anti-Hib PS IgG2, 1.01 for anti-Hib PS IgG3, and 1.09 for anti-Hib PS IgG4. The sera from Korean children of almost all age groups reacted to Hib-PS antigen on ELISA. Also the active transport of anti-Hib PS IgG antibody through placenta was observed. Among four IgG subclasses, only IgG1 transport had significant experimental meaning.

Production of monoclonal antibodies to Vi polysaccharide antigen of Salmonella typhi.

Twenty-four Vi antigen-specific monoclonal antibodies were produced in this study. The MAbs were found to be highly specific to Vi possessing bacteria. Selected MAbs were used in a direct agglutination assay for rapid identification of S. typhi in primary bacterial culture and also used to develop an assay to detect Vi antigen in clinical specimens. The result showed that they could not detect the antigen in urine and serum from acute patients even they could detect as low as 0.02 micrograms/ml of Vi antigen added in normal urine. The study has shown that these MAbs are very useful for rapid identification of S. typhi in primary bacterial culture and they can replace polyclonal anti-Vi antibodies which have been used routinely in bacteriological laboratories.

Polyvalent pneumococcal polysaccharide vaccines: a review of literature

Streptococcus pneumoniae e um agente bacteriano responsavel por uma grande incidencia de morbidade e mortalidade em criancas e em idosos e tambem em individuos portadores do virus da imunodeficiencia humana (HIV). S. pneumoniae tem sido um agente bacteriano isolado com grande frequencia em pneumonias, otite media, sinusite, bacteremia e meningite. A vacina polivalente pneumococica contem os polissacarides capsulares dos 23 sorotipos de S. pneumoniae mais frequentemente encontrados como causadores de infeccoes pneumococicas em diversos paises. Public Health Service Advisory Committee on Imunization Pratice, pertencente ao Centers for Disease Control (CDC), nao recomenda o emprego da vacina pneumococica para a imunizacao da populacao em geral. Contudo, diversos pesquisadores tem recomendado seu emprego para populacoes especiais como por exemplo, os portadores de infeccao por HIV, que estao sujeitos a um alto risco de infeccoes pneumococicas sistemicas. A presente revisao da literatura tem como objetivo divulgar a importancia da vacina polivalente pneumococica bem como suas propriedades imunologicas e seus efeitos na populacao vacinada.

Deteccion de antigeno del polisacarido capsular de Cryptococcus neoformans en pacientes con SIDA y neurocriptococosis en Sao Paulo, Brasil / Detection of capsular polysaccharide antigen of Cryptococcus neoformans in patients with AIDS and neurocryptococcosis in Sao Paulo, Brazil

Antigeno del polisacarido capsular (AgPC) de Cryptococcus neoformans fue detectado por la tecnica de aglutinacion de latex (AL) en LCR y suero de pacientes con Sindrome de Inmunodeficiencia Adquirida (SIDA) y primer episodio de neurocriptococosis, usando como patron el examen micologico (examen directo y cultivo) de LCR. Se obtuvo una sensibilidad de 100 por ciento de AL para detectar AgPC de C. neoformans, el cual por su rapidez permite tratamiento especifico precoz. Titulos iniciales de AgPC de la levadura en esos pacientes pueden ser>1.000.000, pareciendo que cuando esos titulos estan presentes en suero, se relacionan con mortalidad durante el tratamiento. En los pacientes que sobrevivieron se observo que el examen micologico directo y AgPC de C. neoformans, en LCR y suero, permanecen positivos aun despues de tratamiento y mejoria clinica del paciente.

Polisacáridos capsulares como vacunas antibacterianas / Capsular polysaccharide as anti-bacterial vaccines

Many bacterial pathogens are encapsulated, and these capsules are known or thought to be important in their virulence. Capsular polysaccharides are known to be good immunogens, and the first multivalent vaccines based on the capsular polysaccharides of Streptococcus pneumoniae was developed in 1945. However, it was not until recent renewed interst in the prophylaxis of bacterial disease that these materials were reconsidered as vaccine candidates. They offer the advantege of being defined molecular species that can be readily isolated, are non-toxi, and with few exceptions, inmunogenic. Several problems are still associated with polkysaccharide vaccines: a) the poor immunogenicity of all the polysaccharides in infantes, and b) the poor immunogenicity of all the polusaccharides in infants. and b) the poor immunogenicity of a few polusaccharides in al humans. New strategies have been developed in order to overcome these problems, such as chemical modification of the polysaccharides and conjugation of the polysaccharides to carrier proteins. The purpose of this paper is to describe briefly the strutural characteristics of the capsular polysaccharides and their relation to immunogenicity, and be development of vaccines based on capsular polysaccharides with examples of those currently being used and those having future potential use. As well, some of the strategies directed towards producing plysaccharide-protein conjugates will be discussed