DESARROLLO DE UNA METODOLOGÍA POR CROMATOGRAFÍA DE EXCLUSIÓN MOLECULAR PARA LA DETERMINACIÓN DE POLIRRIBOSIL-RIBITOL-FOSFATO (PRP) LIBRE Y CONJUGADO A TOXOIDE TETÁNICO (PRP-TTD) EN VACUNAS ANTI HAEMOPHILUS INFLUENZAE TIPO B / Development of a size exclusion chromatography method for a determination of free polyribosyl-ribitol-phosphate (PRP) and conjugated to tetanus toxoid (PRP-TTD) in Haemophilus influenzae type B vaccines

Haemophilus influenzae tipo b es un patógeno oportunista responsable de infecciones invasivas y graves como bacteriemia y meningitis bacteriana aguda en poblaciones susceptibles, principalmente en niños menores de 5 años de edad. Su antígeno capsular, el polirribosil-ribitol-fosfato, es un polisacárido específico del serotipo b y ha sido utilizado para la producción de vacunas, inicialmente en forma aislada, con escaso éxito debido a la pobre estimulación inmune en la población objetivo, dando una protección poco sostenida en el tiempo. Surgieron así las vacunas conjugadas, usadas actualmente, donde el polisacárido capsular se une a una proteína carrier para generar una respuesta inmune timo-dependiente, con memoria inmunológica, y apta para el uso en niños a partir de los 2 meses de edad. El polirribosil-ribitol-fosfato se caracteriza por ser una secuencia polimérica del azúcar fosfatado de largo variable, formando una mezcla con diferente abundancia de pesos moleculares. El largo de las cadenas polisacarídicas es un parámetro de calidad del proceso productivo de estas vacunas, y se controla mediante técnicas de cromatografía de exclusión molecular. El presente trabajo consistió en desarrollar una técnica analítica para la determinación de polirribosil-ribitol-fosfato en su forma libre, y conjugado a toxoide tetánico, presentes en vacunas anti Haemophilus influenzae tipo b, utilizando cromatografía de exclusión molecular acoplada a detectores por índice de refracción y por luz ultravioleta a longitudes de onda de 215 y 280 nm. El desarrollo se planteó sobre antecedentes bibliográficos que describen una metodología similar como control de la instancia de conjugación en el proceso productivo del ingrediente farmacéutico activo de vacunas anti Haemophilus influenzae tipo b. En el procedimiento se utilizaron estándares internacionales de los analitos libres y estándares de pesos moleculares, y se evaluó la capacidad del sistema para discriminar los componentes de la vacuna y detectar diferencias que podrían indicar un compromiso en la calidad de la misma.

Haemophilus influenzae type b is an opportunistic pathogen responsible for invasive and severe infections such as bacteremia and acute bacterial meningitis in susceptible populations, mainly children under 5 years of age. Its capsular antigen, polyribosylribitol-phosphate, is a specific polysaccharide of serotype b and has been used to produce vaccines, initially in isolation, with little success due to poor immune stimulation in the target population, and providing little sustained protection over time. Thus, arose the conjugated vaccines currently used, where the capsular polysaccharide binds to a carrier protein to generate a thymusdependent immune response with immunological memory and suitable for use in children from 2 months of age. Polyribosyl-ribitolphosphate is characterized by being a polymeric sequence of the phosphated sugar of variable length, forming a mixture with Emilia Ojeda Zachara*, Gisela Severino*, Ana Carolina Abba*, Patricia Aprea. Departamento del Laboratorio Nacional para el Estudio y Control de Biológicos, Dirección de Evaluación y Control de Biológicos y Radiofármacos, Instituto Nacional de Medicamentos. Administración Nacional de Medicamentos, Alimentos y Tecnología Médica, Buenos Aires. Argentina. Correspondencia: Emilia Ojeda Zachara (emilia.ojeda@anmat.gob.ar) *Estos autores contribuyeron equitativamente al desarrollo de este trabajo. Recibido: 26 de julio de 2021. Aprobado: 1 de diciembre de 2021. Revista Científica ANMAT. Año 5 (vol 2), 2021 different abundances of various molecular weights. The length of the polysaccharide chains is a quality parameter of the production process of these vaccines, and is controlled by size-exclusion chromatography techniques. The presented work consisted in developing an analytical technique for the determination of polyribosyl-ribitol-phosphate in its free form and conjugated to tetanus toxoid present in vaccines against Haemophilus influenzae type b, using size-exclusion chromatography coupled to refractive index and UV detectors at wavelengths of 215 and 280 nm. The development was based on bibliographic backgrounds that use a similar methodology as a control of the conjugation step in the production process of the active pharmaceutical ingredient of anti-Haemophilus influenzae type b vaccines. In the procedure, international standards for free analytes and molecular weight standards were used, and the ability of the system to discriminate the vaccine components and to detect differences that could indicate a compromise in the quality of this vaccine was evaluated

Economic Evaluation and Budget Impact Analysis of Vaccination against Haemophilus influenzae Type b Infection in Thailand.

Current study aimed to estimate clinical and economic outcomes of providing the Haemophilus influenzae type b (Hib) vaccination as a national vaccine immunization program in Thailand. A decision tree combined with Markov model was developed to simulate relevant costs and health outcomes covering lifetime horizon in societal and health care payer perspectives. This analysis considered children aged under 5 years old whom preventive vaccine of Hib infection are indicated. Two combined Hib vaccination schedules were considered: three-dose series (3 + 0) and three-dose series plus a booster does (3 + 1) compared with no vaccination. Budget impact analysis was also performed under Thai government perspective. The outcomes were reported as Hib-infected cases averted and incremental cost-effectiveness ratios (ICERs) in 2014 Thai baht (THB) ($) per quality-adjusted life year (QALY) gained. In base-case scenario, the model estimates that 3,960 infected cases, 59 disability cases, and 97 deaths can be prevented by national Hib vaccination program. The ICER for 3 + 0 schedule was THB 1,099 ($34) per QALY gained under societal perspective. The model was sensitive to pneumonia incidence among aged under 5 years old and direct non-medical care cost per episode of Hib pneumonia. Hib vaccination is very cost-effective in the Thai context. The budget impact analysis showed that Thai government needed to invest an additional budget of 110 ($3.4) million to implement Hib vaccination program. Policy makers should consider our findings for adopting this vaccine into national immunization program.

A high throughput serum bactericidal assay for antibodies to Haemophilus influenzae type b.

BACKGROUND: The protective capacities of antibodies induced with Haemophilus influenzae type b (Hib) vaccines can be directly assessed in vitro with a Hib-specific serum bactericidal assay (SBA). However, the conventional SBA requires several tedious steps including manual counting of bacterial colonies, and therefore, it is seldom used. METHODS: To overcome these limitations, we have improved the conventional SBA by using frozen target bacteria and by developing an automated colony counting method based on agar plates with the chromogenic dye 2, 3, 5-triphenyl tetrazolium chloride (TTC). RESULTS: These changes enabled us to analyze about 100 serum samples per day per person by SBA. When the intra- and inter-assay precisions were studied, this assay showed a coefficient of variation (CV) ranging from 1 to 38 %. To monitor the long term assay stability for assays involving different bacteria lots, complement lots, and operators, we analyzed bactericidal indices of quality control samples obtained over a 6 year period and found the CV to be about 35-50 %. Lastly, our SBA results were compared with the ELISA results obtained using 90 serum samples from children. We showed that the bactericidal index correlated with IgG anti-Hib antibody levels (r = 0.84), with a bactericidal index of 10 corresponding approximately to 0.15 µg/mL IgG, the widely accepted protective level of antibody. CONCLUSION: We describe a simple high throughput SBA for anti-Hib antibodies that would be useful for evaluating various Hib vaccines. While additional work will be needed to standardize the assay, this SBA should greatly facilitate studies of Hib vaccines.

Vaccination in Southeast Asia--reducing meningitis, sepsis and pneumonia with new and existing vaccines.

Streptococcus pneumoniae, Haemophilus influenzae type b and Neisseria meningitidis are leading causes of vaccine-preventable diseases such as meningitis, sepsis and pneumonia. Although there has been much progress in the introduction of vaccines against these pathogens, access to vaccines remains elusive in some countries. This review highlights the current S. pneumoniae, H. influenzae type b, and N. meningitidis immunization schedules in the 10 countries belonging to the Association of Southeast Asian Nations (ASEAN). Epidemiologic studies may be useful for informing vaccine policy in these countries, particularly when determining the cost-effectiveness of introducing new vaccines.

High cost is the primary barrier reported by physicians who prescribe vaccines not included in India's Universal Immunization Program.

Haemophilus influenzae type B (Hib) vaccine, pneumococcal conjugate vaccine (PCV) and rotavirus (RV) vaccine are available in the private market in India, but, except for Hib in eight states, are not included in India's Universal Immunization Program (UIP). Pediatricians were surveyed about administering non-UIP vaccines. Most give these vaccines to some of their patients (73-83%, depending on vaccine), but few give them to all patients (7-18%). High cost was the most frequently cited barrier (93-96%). Only 10-12% of respondents had concerns about the efficacy of PCV or RV vaccine, and concerns about Hib vaccine efficacy or any vaccine safety issues were rare (1-3%). Practice varied by type of healthcare facility, with pediatricians at government hospitals least likely to administer non-UIP vaccines. Support for the inclusion of all three in the UIP was high (83-95%). Including Hib vaccine, PCV and RV vaccine in India's UIP would be supported by pediatricians and help eliminate the current barrier of high cost of these immunizations.

A fully liquid DTPw-HepB-Hib combination vaccine for booster vaccination of toddlers in El Salvador / Vacuna líquida combinada DTPw-HepB-Hib como dosis de refuerzo en infantes de El Salvador

Objectives:To compare the safety and immunogenicity of a booster dose of a fully liquid diphtheria-tetanus-whole cell pertussis-hepatitis B-Haemophilus influenzae type b (DTPw-HepB-Hib) vaccine to the separate administration of commercially available DTPw and Hib vaccines in healthy toddlers. Methods:An open-label, randomized, parallel-group, Phase III study conducted at six centers in San Salvador, El Salvador, during February-June 2006. Toddlers (15-24 months of age) were eligible to participate if they had received primary immunization at 2, 4, and 6 months of age with a commercial DTPw-HepB/Hib vaccine requiring reconstitution. Participants received either one booster dose of DTPw-HepB-Hib fully liquid vaccine or DTPw and Hib vaccines administered separately. Blood samples were taken immediately prior to and at 1 month post-vaccination. For a 5-day period following vaccination, solicited adverse events were collected in subject diaries and assessed. Results:The combined DTPw-HepB-Hib fully liquid vaccine was non-inferior to the separately administered DTPw and Hib vaccines, in terms of seroprotection/seroconversion rates for all antigens evaluated. The combination vaccine elicited a strong booster response as demonstrated by a large increase in antibodies against all vaccine antigens. The geometric mean concentrations (GMCs) of all antibodies in the DTPw-HepB-Hib group exceeded the seroprotection/seroconversion thresholds by very large margins, although for some antigens they were somewhat lower than the corresponding titers in the comparator group. With the combination vaccine, considerably fewer solicited local and systemic adverse events, such as fever and irritability, were reported than with the comparator vaccines. Conclusions:This study demonstrates that the fully liquid combined DTPw-HepB-Hib vaccine is highly immunogenic and has a favorable safety profile when given as a booster vaccination to toddlers who have received...

Objetivos:Comparar la seguridad y la inmunogenicidad en infantes saludables de una dosis de refuerzo de una vacuna líquida combinada contra la difteria, el tétanos, la tosferina (de células enteras), la hepatitis B y Haemophilus influenzae tipo b (DTPw-HepB-Hib), con la aplicación por separado de vacunas DTPw y Hib disponibles comercialmente. Métodos:Se realizó un estudio de fase III abierto, aleatorizado, con grupos paralelos, en seis centros de San Salvador, El Salvador, en febrero-junio de 2006. Los infantes (de 15-24 meses) habían recibido la inmunización primaria a los 2, 4 y 6 meses de edad con una vacuna comercial DTPw-HepB/Hib que necesitaba reconstitución. Los lactantes recibieron una dosis de refuerzo con la vacuna DTPw-HepB-Hib o las vacunas DTPw y Hib por separado. Se tomaron muestras de sangre inmediatamente antes de la vacunación y un mes después. Las reacciones adversas en los cinco días siguientes a la vacunación se anotaron en diarios individuales y se evaluaron. Resultados:Según las tasas de seroprotección/seroconversión de todos los antígenos evaluados, la vacuna DTPw-HepB-Hib no fue inferior que las vacunas DTPw y Hib administradas por separado. La vacuna combinada produjo una fuerte respuesta de refuerzo, reflejada en el gran aumento de anticuerpos contra todos los antígenos presentes. Con respecto al grupo de comparación, en el grupo vacunado con DTPw-HepB-Hib las concentraciones geométricas medias de todos los anticuerpos superaron ampliamente los umbrales de seroprotección/seroconversión -aunque con títulos menores en algunos antígenos- y hubo mucho menos reacciones adversas locales y sistémicas, como fiebre e irritabilidad. Conclusiones:Se demostró que la vacuna líquida combinada DTPw-HepB-Hib es altamente inmunógena y satisfactoriamente segura cuando se aplica como dosis de refuerzo a infantes inmunizados primariamente con una vacuna pentavalente diferente que requiere reconstitución.

Burden of Haemophilus influenzae type B disease in Pakistani children

Estimates of the burden of Haemophilus influenzae type b [Hib] in children in Pakistan are limited. A prospective surveillance was set up in 8 sentinel sites in Karachi and Hyderabad in January 2004. A total of 1481 children aged < 5 years underwent lumbar puncture for suspected acute bacterial meningitis. Specimens from 237 [16.0%] children met the criteria for probable bacterial meningitis, and Hib was detected in 45 of them [19.0%]. The minimum detected incidence of Hib meningitis in the Hyderabad area was 7.6 per 100 000 in children < 5 years of age, and 38.1 per 100 000 children < 1 year. Hib vaccination is justified for inclusion in Pakistan's expanded programme of immunization

Prevalence of oropharyngeal colonization by Haemophilus influenzae type b in Iranian children

Healthy carriers of Haemophilus influenzae type b [Hib] play an important role in the spread of invasive disease. The aim of this study was to assess the need for Hib vaccination in Iranian children by estimating the prevalence of Hib oropharyngeal colonization among children in Tehran. Cultures were prepared from oropharyngeal swabs of 1000 children in 25 day-care centres in Tehran from October 2005 to March 2006. The prevalence of Hib carriers was 7.6%, similar to other developing countries prior to inoculation with the conjugate Hib vaccine. We recommend Hib vaccination be included in the Iranian national programme of immunization

Haemophilus influenzae type b diseases in children: a pre-vaccination study

Haemophilus influenzae type b [Hib] can now be prevented by vaccination. We present the clinical and laboratory characteristics of acute invasive H. influenzae diseases in children admitted over a 4-year period to a tertiary paediatric ward of the Al-Ain medical district hospital, before vaccination became available in the United Arab Emirates. In all, 38 children had bacteriologically proven H. influenzae invasive diseases and all the isolates were serotype b. Meningitis was diagnosed in 60.5% of the children and 66% of the studied children were under 12 months. There were no deaths but substantial morbidity occurred in 12 children

Vaccines: World Health Organization versus Federal Drug Administration recommended formula

Vaccines produced in accordance with WHO formulas, differ in concentration from those used in United States according to FDA formulas. We aimed to compare the immunogenicity of both formulas. Infants who were 6 weeks old were randomly put into 3 groups to receive 3 doses of vaccines at 6 weeks, 3 months and 5 months of age. The vaccines consisted of Haemophilus influenzae type b vaccine, diphtheria-tetanus-pertussis and oral polio vaccine. Antibody levels for polyribosylribitol phosphate [PRP], tetanus, diphtheria and poliovirus were measured 1 month after the third dose of vaccines. Although diphtheria and tetanus antigens in the FDA formula are half the concentration of the WHO formula, anti-tetanus and anti-diphtheria antibodies were significantly higher. No difference was found between groups regarding oral poliovirus vaccine