Evaluación neuroepidemiológica a partir de anticuerpos antivirales de memoria generados por la vacuna triple viral / Neuro-Epidemiological Evaluation From Memory Antiviral Antibodies Generated By The MMR Vaccine

Introducción: La vacuna contra sarampión-parotiditis-rubéola es administrada en Cuba con un 99 por ciento de cobertura vacunal. Actualmente se plantea la baja inmunogenicidad de la cepa de parotiditis con que se fabrica por lo que resulta importante velar por la eficacia de las vacunas y su respuesta inmune protectora. Objetivos: Evaluar el comportamiento de los índices de anticuerpo antirubéola, antiparotiditis y antisarampión de acuerdo con la edad, y analizar la variación de la respuesta de anticuerpos antiparotiditis con respecto a estudios anteriores. Materiales y métodos: Se estudiaron muestras de suero y líquido cefalorraquídeo de 42 pacientes pediátricos con procesos neuroinflamatorios y se les cuantificó IgG total y albúmina y anticuerpos específicos contra los tres virus a partir de ensayos innmunoenzimáticos tipo ELISA. Se realizaron los reibergramas correspondientes e índices de anticuerpos específicos. Resultados: Se observó un incremento sostenido de anticuerpos contra los tres inmunógenos de forma general, sin diferencias significativas por razones de edad ni cambios notables posrevacunación. Existió un decrecimiento del índice de anticuerpos a medida que se alejaba de la fecha de revacunación por lo que se debe mantener una vigilancia en esos grupos de edades. La velocidad de producción de anticuerpos antiparotiditis fue mayor que frente a los otros virus, aunque no de forma significativa. Conclusiones: Esta vacuna garantiza protección por la uniformidad de la respuesta inmune de memoria inducida en todos los grupos de edades. Se demostró un aumento de protección de la población estudiada frente a la parotiditis con respecto a estudios previos(AU)

Introduction: The measles-mumps-rubella vaccine is administered in Cuba with 99 percent vaccination coverage. Currently, the low immunogenicity of the strain of mumps with which it is manufactured is raised, so it is important to ensure the effectiveness of vaccines and their protective immune response. Objectives: Evaluate the behavior of the anti-rubella, anti-mumps and anti-measles antibody indices according to age, and to analyze the variation of the anti-mumps antibody response with respect to previous studies. Materials and methods: Serum and cerebro-spinal fluid samples from 42 pediatric patients with neuro-inflammatory processes were studied and total IgG and albumin and specific antibodies against the three viruses were quantified from immunoenzymatic assays ELISA type. Corresponding reibergrams and specific antibody indices were performed. Results: A sustained increase in antibodies against the ethree immunogens was observed in general, without significant differences due to age or notable post-vaccination changes. There was a decrease in the antibody index as it moved away from the date of revaccination, so surveillance should be maintained in these age groups. The rate of production of anti-mumps antibodies was higher than against the other viruses, although not significantly. Conclusions: This vaccine guarantees protection by the uniformity of the memory induced immune response in all age groups. An increase in the protection of the studied population against mumps was demonstrated with respect to previous studies(AU)

Kinetic Characteristics of Neutralizing Antibody Responses Vary among Patients with COVID-19 / 生物医学与环境科学（英文）

Objective@#The coronavirus disease 2019 (COVID-19) pandemic continues to present a major challenge to public health. Vaccine development requires an understanding of the kinetics of neutralizing antibody (NAb) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).@*Methods@#In total, 605 serum samples from 125 COVID-19 patients (from January 1 to March 14, 2020) varying in age, sex, severity of symptoms, and presence of underlying diseases were collected, and antibody titers were measured using a micro-neutralization assay with wild-type SARS-CoV-2.@*Results@#NAbs were detectable approximately 10 days post-onset (dpo) of symptoms and peaked at approximately 20 dpo. The NAb levels were slightly higher in young males and severe cases, while no significant difference was observed for the other classifications. In follow-up cases, the NAb titer had increased or stabilized in 18 cases, whereas it had decreased in 26 cases, and in one case NAbs were undetectable at the end of our observation. Although a decreasing trend in NAb titer was observed in many cases, the NAb level was generally still protective.@*Conclusion@#We demonstrated that NAb levels vary among all categories of COVID-19 patients. Long-term studies are needed to determine the longevity and protective efficiency of NAbs induced by SARS-CoV-2.

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab’)2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab’)2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.

Epitope of dengue virus E protein detect human antibodies associated with mild disease: a potential peptide for vaccine development

ABSTRACT The antigenic potential of seven immunogenic peptides of the dengue virus was evaluated in the sera of patients with dengue confirmed by IgM/IgG serology. Antibodies IgM and IgG against dengue virus peptides were analyzed by ELISA in 31 dengue sero-positive and 20 sero-negative patients. The P5 peptide showed significant IgG immunoreactivity mostly in the sera of patients with dengue without warning signs in comparison with patients with dengue with warning signs, correlating with mild disease. This finding suggests that the low antibody response against P5 epitope could be a risk factor for higher susceptibility to dengue virus infection with warning signs, and that P5 could be a potential antigen for vaccine development.

The direct and indirect effects of the pneumococcal conjugated vaccine on carriage rates in children aged younger than 5 years in Latin America and the Caribbean: a systematic review / Efeitos diretos e indiretos da vacina pneumocócica conjugada no estado de portador em crianças menores de 5 anos, na América Latina e no Caribe: uma revisão sistemática

ABSTRACT Objective To demonstrate the impact of pneumococcal conjugate vaccine in Streptococcus pneumoniae carriage status in children younger than 5 years in Latin America and the Caribbean. Methods A systematic literature review was carried out on the direct and indirect effects of pneumococcal vaccine in the carriage status, after implementation in childhood immunization programs. Studies carried out in children younger than 5 years were selected from the PubMed® and Virtual Health Library databases, and data collected after implementation of pneumococcal vaccine in Latin America and the Caribbean, between 2008 and 2018. Results From 1,396 articles identified, 738 were selected based on titles and abstracts. After duplicate removal, 31 studies were eligible for full-text reading, resulting in 6 publications for analysis. All selected publications were observational studies and indicated a decrease in the carriage and vaccine types, and an increase in the circulation of non-vaccine serotypes, such as 6A, 19A, 35B, 21 and 38. We did not identify changes in the antimicrobial resistance after vaccine implementation. Conclusion A decrease in the carriage status of vaccine types and non-vaccine types was detected. The continuous monitoring of pneumococcal vaccine effect is fundamental to demonstrate the impact of the carriage status and, consequently, of invasive pneumococcal disease, allowing better targeting approaches in countries that included pneumococcal vaccine in their immunization programs. Our study protocol was registered in PROSPERO (www.crd.york.ac.uk/prospero) under number CRD42018096719.

RESUMO Objetivo Demonstrar o impacto das vacinas pneumocócicas conjugadas no estado de portador de Streptococcus pneumoniae em crianças menores de 5 anos na América Latina e no Caribe. Métodos Foi realizada revisão sistemática da literatura sobre os efeitos diretos e indiretos da vacina pneumocócica no estado de portador em crianças menores de 5 anos, após a implantação da vacina nos calendários de imunização infantil. A partir de dados da PubMed®e da Biblioteca Virtual da Saúde, foram selecionados estudos de portador em crianças menores de 5 anos, com dados coletados após implementação da vacina de 2008 a 2018, na América Latina e no Caribe. Resultados Dos 1.396 artigos identificados, 738 foram selecionados mediante leitura de títulos e resumos. Após a extração dos duplicados, 31 foram elegíveis para leitura do texto completo, restando 6 artigos para análise. Todos os estudos selecionados eram observacionais e indicavam diminuição do portador e tipos vacinais, e aumento da circulação de sorotipos não vacinais, como 6A, 19A, 35B, 21 e 38. Não foi observada alteração na resistência antimicrobiana após a introdução da vacina. Conclusão Detectou-se redução no estado de portador, dos tipos vacinais e não vacinais. O monitoramento contínuo do efeito das vacinas pneumocócicas é fundamental, para demonstrar o impacto do estado de portador e, consequentemente, da doença pneumocócica invasiva, permitindo o melhor direcionamento nas ações em saúde para os países que incluíram a vacina no calendário de imunização. Nosso protocolo de estudo foi registrado no PROSPERO (www.crd.york.ac.uk/prospero) sob o número CRD42018096719.

Performance evaluation of an indirect immunofluorescence kit for the serological diagnosis of dengue / Avaliação do desempenho de kit de imunofluorescência indireta para o diagnóstico sorológico de dengue

ABSTRACT Objective: To evaluate the performance of indirect immunofluorescence for serological diagnosis of dengue virus in a population with high prevalence of arboviruses. Methods: Two-hundred serum samples from patients with clinical suspicion of dengue fever were tested by immunoenzymatic and indirect immunofluorescence assay BIOCHIP® mosaic. Specificity, sensitivity and Kappa coefficient were calculated. Discordant samples were tested by polymerase chain reaction for confirmation. Results: Of the 200 samples, 20% were positive and 80% negative for anti-dengue virus IgM antibodies in the immunoenzymatic test. Of the 40 positives, 25% were negative in indirect immunofluorescence. Of these ten discordant results, only 20% were also negative in the polymerase chain reaction (PCR). Of the 160 negatives in the immunoenzymatic test, 5% were positive in indirect immunofluorescence. Of these nine discordant results, 33% were positive in the PCR. The Kappa coefficient was 0.7 (0.572-0.829). Sensitivity and specificity of indirect immunofluorescence were respectively 75% and 94%. For anti-dengue virus IgG antibodies, of the 200 samples, 15.5% were positive and 84.5% were negative in the immunoenzymatic test. Of the 31 positives, 12.9% were negative in indirect immunofluorescence. Of these four discordant results, 25% were negative in the PCR. Of the 169 negatives, 8% were positive in indirect immunofluorescence. Of these 14 discordant results, 64% were also positive in the PCR. The Kappa coefficient was 0.695 (0.563-0.83). Sensitivity and specificity of indirect immunofluorescence were 87.1% and 91.7%, respectively. Conclusion: For diagnosis of acute infection, the immunoenzymatic test is enough, and the use of additional methods is not warranted. Replacing the immunoenzymatic test by indirect immunofluorescence would compromise the sensitivity for IgM. However, indirect immunofluorescence can distinguish three arboviruses simultaneously, an advantage during concomitant epidemics.

RESUMO Objetivo: Avaliar o desempenho da imunofluorescência indireta no diagnóstico sorológico de dengue em uma população com alta prevalência de arboviroses. Métodos: Duzentas amostras de soro de pacientes com suspeita clínica de dengue foram testadas por ensaio imunoenzimático e imunofluorescência indireta mosaico BIOCHIP®. Foram calculados especificidade, sensibilidade e coeficiente Kappa. Nas amostras discordantes, realizou-se reação em cadeia da polimerase como método confirmatório. Resultados: Das 200 amostras, 20% foram positivas e 80% negativas para IgM antivírus da dengue no ensaio imunoenzimático. Das 40 positivas, 25% foram negativas na imunofluorescência indireta. Destas dez negativas, apenas 20% eram também negativas na reação em cadeia da polimerase. Das 160 negativas no ensaio imunoenzimático, 5% foram positivas na imunofluorescência indireta. Por fim, dentre as nove discordantes, 33% tiveram vírus da dengue detectado na reação em cadeia da polimerase. O coeficiente Kappa foi 0,70 (0,57-0,82). Sensibilidade e especificidade por imunofluorescência indireta foram, respectivamente, 75% e 94%. Para IgG antivírus da dengue, de 200 amostras, 15,5% foram positivas e 84,5% negativas no ensaio imunoenzimático. Das 31 positivas, 12,9% foram negativas na imunofluorescência indireta. Destas quatro discordantes, 25% apresentaram vírus da dengue não detectado na reação em cadeia da polimerase. Das 169 negativas, 8% foram positivas na imunofluorescência indireta. Destas, 64% foram positivas também na reação em cadeia da polimerase. O coeficiente Kappa foi 0,695 (0,56-0,83). Sensibilidade e a especificidade por imunofluorescência indireta foram, respectivamente, 87,1% e 91,7%. Conclusão: Ensaio imunoenzimático seria suficiente para diagnóstico sorológico de infecção aguda, não justificando a incorporação da imunofluorescência indireta. Substituir ensaio imunoenzimático pela imunofluorescência indireta poderia comprometer a sensibilidade para IgM. Contudo, a imunofluorescência indireta auxilia diferenciar três arboviroses simultaneamente, sendo vantajoso em epidemias concomitantes.

Low risk of serological cross-reactivity between dengue and COVID-19

In the near future, the overlap of Coronavirus disease 2019 (COVID-19) and dengue epidemics is a concrete threat in tropical regions. Co-epidemics of COVID-19 and dengue could be an overwhelming challenge for health systems in low- and middle-income countries. In this work, we investigated potential serological cross-reactions between COVID-19 and dengue patients. Among 32 COVID-19 positive sera, no positive Dengue virus (DENV) IgG/IgM results were observed. On the other hand, one false-positive result was observed among 44 DENV-positive sera tested for COVID-19 antibodies with each of the two rapid tests used. Further data on accuracy of COVID-19 diagnostic test are urgently warranted.

Serum regucalcin is a useful indicator of liver injury severity in patients with hepatitis B virus-related liver diseases

Regucalcin is a soluble protein that is principally expressed in hepatocytes. Studies of regucalcin have mainly been conducted in animals due to a lack of commercially available kits. We aimed to develop an enzyme-linked immunosorbent assay (ELISA) to quantify serum regucalcin in patients with hepatitis B virus (HBV)-related disease. High-titer monoclonal antibodies and a polyclonal antibody to regucalcin were produced, a double-antibody sandwich ELISA method was established, and serum regucalcin was determined in 47 chronic hepatitis B (CHB) patients, 91 HBV-related acute-on-chronic liver failure (HBV-ACLF) patients, and 33 healthy controls. The ELISA demonstrated an appropriate linear range, and high levels of reproducibility, sensitivity, specificity, accuracy, and stability. The median serum regucalcin concentrations in HBV-ACLF and CHB patients were 5.46 and 3.76 ng/mL, respectively (P<0.01), which were much higher than in healthy controls (1.72 ng/mL, both P<0.01). For the differentiation of CHB patients and healthy controls, the area under curve (AUC) was 0.86 with a cut-off of 2.42 ng/mL, 85.7% sensitivity, and 78.8% specificity. In contrast, the AUC of alanine aminotransferase (ALT) was lower (AUC=0.80, P=0.01). To differentiate ACLF from CHB, the AUC was 0.72 with a cut-off of 4.26 ng/mL, 77.0% sensitivity, and 61.2% specificity while the AUC of ALT was 0.41 (P=0.07). Thus, we have developed an ELISA that is suitable for measuring serum regucalcin and have shown that serum regucalcin increased with the severity of liver injury due to HBV-related diseases, such that it appears to be more useful than ALT as a marker of liver injury.

Diagnostic significance of immunoglobulin G avidity in symptomatic and asymptomatic West Nile virus infection

Abstract INTRODUCTION West Nile virus (WNV) immunoglobulin M (IgM) antibodies have been shown to persist for up to 500 days in certain patients. To evaluate the usefulness of immunoglobulin G (IgG) avidity assessment in the diagnosis of WNV infection, we analyzed 54 WNV IgM- and/or IgG-positive serum samples from 39 patients with neuroinvasive disease and 15 asymptomatic cases tested during a seroprevalence investigation. METHODS Serological tests (WNV IgM/IgG antibody detection, IgG avidity) were performed using commercially available enzyme-linked immunosorbent assays. RESULTS WNV IgM antibodies were detected in 47 (87%) samples. Acute/recent WNV infection was confirmed based on low/borderline avidity index (AI) in 44 IgM-positive samples (93.6%). In three IgM-positive samples (6.4%), high IgG AIs were detected, thus indicating persisting IgM antibodies from previous infections. All IgM-negative samples showed high AIs. Patients with WNV neuroinvasive disease tested within 30 days showed low AIs. In six patients tested 34-50 days after disease onset, AI was borderline (42%-60%), suggesting earlier WNV IgG maturation. Samples with the highest IgM values were associated with the lowest AIs (Spearman's rho coefficient -0.767, p < 0.001). CONCLUSIONS Our results indicate that IgG avidity differentiates current/recent WNV infection from persistent IgM seropositivity from the previous WNV transmission season both in patients with WNV neuroinvasive disease and in asymptomatic persons. A strong negative correlation between IgM antibody levels and AI indicates that in cases with very high IgM levels, determination of IgG avidity may not be necessary. As many patients showed rapid avidity maturation, low IgG avidity is indicative of WNV infection within the previous month.

Seronegativity to polio viruses among previously immunized adult candidates to solid organ transplantation

ABSTRACT In the current effort to eliminate polio from the world, it is important to recognize and vaccinate susceptible groups, especially immunocompromised patients living in countries where attenuated polio vaccine is still used. In this report, we describe the frequency of protective antibodies in a small sample of adult SOT candidates in whom previous vaccination could be ascertained. Patients included in this report were selected among the participants of an ongoing prospective study carried out at the Reference Center for Special Immunobiologicals of the Evandro Chagas National Institute of Infectious Diseases in Rio de Janeiro, Brazil. Among the first 100 patients enrolled in this study, only seven adult SOT candidates had proven polio vaccination at childhood. Three of these seven patients (43%) had no protective antibody titers to one or more poliovirus subtype before solid organ transplant. Proven childhood vaccination against polio does not reliably provide lifelong protective antibody titers for adult SOT candidates and should not be used as a criterion to analyze the need for vaccination in this population.

Estabilidad de la vacuna Candid#1 para prevenir la fiebre hemorrágica Argentina / Stability of Candid#1 vaccine to prevent Argentine Hemorrhagic Fever

Candid#1 es la primera vacuna a virus vivo atenuado producida y registrada en Argentina. Se produce en el INEVH desde 2003 para prevenir la fiebre hemorrágica argentina y se obtiene mediante cosecha de sobrenadantes de cultivos de células diploides infectadas con una cepa atenuada del virus Junín, formulación y posterior liofilización. Su estabilidad es crucial para asegurar su efectividad. El objetivo de este trabajo fue evaluar la estabilidad de Candid#1 exponiéndola a distintas condiciones de temperatura y tiempo. Tres lotes producidos en 2003 fueron sometidos al siguiente esquema de almacenamiento: (a) vacuna reconstituida conservada entre 2 °C y 8 °C durante 8 días, (b) vacuna liofilizada conservada entre 2 °C y 8 °C durante 6 meses, y (c) vacuna liofilizada conservada entre -18 °C y -20 °C durante 10 años. La potencia fue evaluada en monocapa de células Vero bajo agar. Los resultados fueron: (a) Candid#1 reconstituida fue estable 8 días entre 2 °C y 8 °C, (b) Candid#1 liofilizada fue estable 2 meses entre 2 °C y 8 °C y (c) Candid#1 liofilizada fue estable 9 años entre -18 °C y -20 °C manteniendo todos sus atributos. Estos resultados permitieron establecer las siguientes condiciones de almacenamiento: reconstituida 12 horas entre 2 °C y 8 °C, liofilizada 30 días entre 2 °C y 8 °C y 9 años entre -18 °C y -20 °C. A la luz de estos resultados, se generaron cambios favorables en las condiciones de transporte, almacenamiento y distribución de la vacuna. Se implementó la instalación de freezers domésticos en centros estratégicamente distribuidos, permitiendo preservar stocks de vacuna y distribuir las dosis necesarias a vacunatorios.

Candid#1 is the first live attenuated vaccine produced and registered in Argentina. Produced since 2003 at the INEVH to prevent Argentine hemorrhagic fever, it is obtained by harvesting supernatants of diploid cells infected with an attenuated strain of Junin virus and subsequent lyophilization. The stability of this vaccine is crucial to ensure its effectiveness. This study was aimed to evaluate the stability of Candid#1 by exposing it to different time and temperature conditions. Three vaccine batches produced in 2003 were analysed according to the following storage scheme: (a) reconstituted vaccine at 2 °C to 8°C for 8 days; (b) lyophilized vaccine at 2 °C to 8 °C for 6 months; (c) lyophilized vaccine at -18 °C to -20 °C for 10 years. The potency was assessed in Vero cell monolayers under agar. The results were: (a) reconstituted vaccine was stable between 2 °C and 8 °C for 8 days, (b) lyophilized vaccine was stable between 2 °C and 8 °C for 2 months, and (c) lyophilized vaccine was stable 9 years between -18 °C and -20 °C, keeping all its properties. These results allowed us to establish the following storage conditions and expiration times for Candid#1: (a) reconstituted: 12 hours between 2 °C and 8 °C, (b) lyophilized: 30 days between 2 °C and 8 °C and (c) lyophilized: 9 years between -18 °C and -20 °C. Based on our results, favorable changes were made in the conditions of transport, storage and distribution of the vaccine. Domestic freezers in strategically located centers were installed, allowing the preservation of vaccine stocks for distribution to secondary vaccination centers.

Anticuerpos policlonales contra la proteína recombinante NS3 del virus del dengue / Polyclonal antibodies against recombinant dengue virus NS3 protein

Resumen Introducción: El dengue es una enfermedad causada por uno de los cuatro serotipos del virus del dengue (DENV) y es endémica en, aproximadamente, 130 países. Su incidencia ha aumentado notablemente en las últimas décadas, así como la frecuencia y la magnitud de los brotes. A pesar de los esfuerzos, no existen tratamientos profilácticos ni terapéuticos contra la enfermedad y, en ese contexto, el estudio de los procesos que gobiernan el ciclo de infección del DENV es esencial para desarrollar vacunas o terapias antivirales. Una de las moléculas del DENV más prometedoras es la proteína no estructural 3 (NS3), la cual es indispensable para la replicación viral y es uno de los principales blancos inmunológicos durante la infección. Objetivo: Producir anticuerpos policlonales para contribuir a los futuros estudios sobre las interacciones entre la proteína NS3 y otras proteínas celulares. Materiales y métodos: Se expresaron dos proteínas recombinantes del dominio helicasa de NS3 del DENV de serotipo 2, las cuales se emplearon para inmunizar ratas y producir anticuerpos policlonales. Resultados: Los anticuerpos producidos fueron útiles en ensayos de Western blot e inmunofluorescencia y se reportó por primera vez un anticuerpo policlonal anti-NS3 que permitió la inmunoprecipitación de la proteína viral y la detecta con Western blot sin necesidad de inducir sobreexpresión de NS3 o de usar extractos de células marcados metabólicamente con radioisótopos. Conclusión: Las proteínas recombinantes expresadas y los anticuerpos producidos constituyen herramientas valiosas para estudiar procesos infecciosos del DENV que involucren a la proteína NS3 y evaluar pruebas dirigidas a interferir las funciones de esta proteína.

Abstract Introduction: Dengue is a disease caused by one of four serotypes of the dengue virus (DENV) and is endemic in approximately 130 countries. The incidence of dengue has increased dramatically in recent decades, as well as the frequency and magnitude of outbreaks. Despite all efforts, there are no prophylactic or therapeutic treatments for the disease. Accordingly, research on the processes governing the DENV infection cycle is essential to develop vaccines or antiviral therapies. One of the most attractive DENV molecules to investigate is nonstructural protein 3 (NS3), which is essential for viral replication and a major immune target for infection. Objective: To produce antibodies to support future studies on NS3 and its cellular interactions with other proteins. Materials and methods: Two recombinant proteins of the helicase domain of DENV NS3 serotype 2 were expressed, and used to immunize mice and produce polyclonal antibodies. Results: The antibodies produced were useful in Western blot and immunofluorescence tests. We report an NS3 antibody that immunoprecipitates the viral protein and detects it in Western blot with no need to over-express it or use cell extracts with metabolic radiolabeling.

Comparative study of lymphocytes from individuals that were vaccinated and unvaccinated against the pandemic 2009-2011 H1N1 influenza virus in Southern Brazil

ABSTRACTINTRODUCTION:While no single factor is sufficient to guarantee the success of influenza vaccine programs, knowledge of the levels of immunity in local populations is critical. Here, we analyzed influenza immunity in a population from Southern Brazil, a region with weather conditions that are distinct from those in the rest of country, where influenza infections are endemic, and where greater than 50% of the population is vaccinated annually.METHODS:Peripheral blood mononuclear cells were isolated from 40 individuals. Of these, 20 had received the H1N1 vaccine, while the remaining 20 were unvaccinated against the disease. Cells were stimulated in vitro with the trivalent post-pandemic influenza vaccine or with conserved major histocompatibility complex I (MHC I) peptides derived from hemagglutinin and neuraminidase. Cell viability was then analyzed by [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide)]-based colorimetric assay (MTT), and culture supernatants were assayed for helper T type 1 (Th1) and Th2-specific cytokine levels.RESULTS:Peripheral blood lymphocytes from vaccinated, but not unvaccinated, individuals exhibited significant proliferation in vitro in the presence of a cognate influenza antigen. After culturing with vaccine antigens, cells from vaccinated individuals produced similar levels of interleukin (IL)-10 and interferon (IFN)-γ, while those from unvaccinated individuals produced higher levels of IFN-γ than of IL-10.CONCLUSIONS:Our data indicate that peripheral blood lymphocytes from vaccinated individuals are stimulated upon encountering a cognate antigen, but did not support the hypothesis that cross-reactive responses related to previous infections can ameliorate the immune response. Moreover, monitoring IL-10 production in vaccinated individuals could comprise a valuable tool for predicting disease evolution.

Development of a novel plaque reduction neutralisation test for hantavirus infection

In the Americas, hantaviruses cause severe cardiopulmonary syndrome (HCPS) with a high fatality rate. Hantavirus infection is commonly diagnosed using serologic techniques and reverse transcription-polymerase chain reaction. This paper presents a novel plaque reduction neutralisation test (PRNT) for detecting antibodies to Brazilian hantavirus. Using PRNT, plaque detection was enhanced by adding 0.6% of dimethyl sulfoxide into the overlay culture medium of the infected cells. This procedure facilitated clear visualisation of small plaques under the microscope and provided for easy and accurate plaque counting. The sera from 37 HCPS patients from the city of Ribeirão Preto, Brazil was evaluated for the Rio Mamoré virus (RIOMV) using PRNT. Six samples exhibited neutralising antibodies; these antibodies exhibited a low titre. The low level of seropositive samples may be due to fewer cross-reactions between two different hantavirus species; the patients were likely infected by Araraquara virus (a virus that has not been isolated) and RIOMV was used for the test. This assay offers a new approach to evaluating and measuring neutralising antibodies produced during hantavirus infections and it can be adapted to other hantaviruses, including viruses that will be isolated in the future.

Internações sensíveis à atenção primária específicas de mulheres / Sensitive female-specific hospitalization in primary care

Objetivou-se analisar as internações por condições sensíveis à atenção primária (ICSAP) específicas em mulheres e os fatores que determinam ou influenciam a ocorrência dessas internações (fatores socioeconômicos, sociodemográficos e controle de saúde) por meio de um inquérito de morbidade hospitalar realizado com amostra de 429 mulheres internadas em hospitais conveniados ao Sistema Único de Saúde. O percentual de ICSAP foi 49,42% (n = 212), com destaque para as internações específicas do sexo feminino 19,35% (n = 83). Associaram ao risco de internar por CSAP: idade superior a 60 anos, baixa escolaridade, internação prévia, realização de controle regular de saúde, falta de vínculo com a Estratégia Saúde da Família (ESF) e ser gestante. As causas evidentes foram as condições relacionadas à gravidez, ao parto e ao puerpério e às inflamações nos órgãos pélvicos femininos. Os resultados sugerem falhas no atendimento ambulatorial que deveria ser oportuno e resolutivo no contexto da saúde da mulher.

The scope of this paper was to analyze female-specific sensitive hospitalization occurring in primary care conditions and factors that determine or affect the occurrence of such hospitalizations (social, economic and demographic factors; health control). Analysis was performed by surveys on hospital morbidity with a sample of 429 females attended in Unified Health System (SUS) contracted hospitals. The sensitive hospitalizations percentage in primary care reached 49.42% (n = 212), highlighting female-specific hospitalization at 19.35% (n = 83). Hospitalization risks comprised elderly people over sixty, low schooling, previous hospitalizations, normal health control, lack of association with the Family Health Strategy and pregnancy. Evident causes were related to conditions of pregnancy, childbirth, post-partum and inflammations of the female pelvic organs. Results suggested flaws in outpatient attendance that should be adequate and provide solutions in women’s health.

Dengue vaccines: what we know, what has been done, but what does the future hold? / Vacinas contra a dengue: o que sabemos, o que tem sido feito, mas o que nos reserva o futuro?

Dengue, a disease caused by any of the four serotypes of dengue viruses, is the most important arthropod-borne viral disease in the world in terms of both morbidity and mortality. The infection by these viruses induces a plethora of clinical manifestations ranging from asymptomatic infections to severe diseases with involvement of several organs. Severe forms of the disease are more frequent in secondary infections by distinct serotypes and, consequently, a dengue vaccine must be tetravalent. Although several approaches have been used on the vaccine development, no vaccine is available against these viruses, especially because of problems on the development of a tetravalent vaccine. Here, we describe briefly the vaccine candidates available and their ability to elicit a protective immune response. We also discuss the problems and possibilities of any of the vaccines in final development stage reaching the market for human use.

Dengue, doença causada por qualquer um dos quatro sorotipos dos vírus dengue, é atualmente a mais importante doença viral transmitida por artrópodos em todo o mundo, tanto em termos de morbidade como de mortalidade. A infecção por estes vírus causa grande variedade de manifestações clínicas, desde infecções assintomáticas até doenças graves com envolvimento de diversos órgãos. As formas graves da dengue são mais frequentes em infecções secundárias por sorotipos diferentes e, por esta razão, a vacina contra a dengue deve ser tetravalente. Embora várias estratégias tenham sido usadas no desenvolvimento de vacinas contra a dengue, não há ainda nenhuma vacina disponível, particularmente por problemas no desenvolvimento de uma vacina tetravalente. Aqui, descreve-se brevemente os candidatos vacinais disponíveis e a capacidade de eles induzirem resposta imune protetora contra novas infecções. Ainda, discutimos os problemas e as possibilidades de liberação, para uso em seres humanos, de qualquer uma das vacinas em fase final de desenvolvimento

Immune response to the mumps component of the MMR vaccine in the routine of immunisation services in the Brazilian National Immunisation Program

A non-controlled longitudinal study was conducted to evaluate the combined vaccine against measles, mumps and rubella (MMR) immunogenicity in 150 children vaccinated in the routine of three health units in the city of Rio de Janeiro, Brazil, 2008-2009, without other vaccines administered during the period from 30 days before to 30 days after vaccination. A previous study conducted in Brazil in 2007, in 1,769 children ranging from 12-15 months of age vaccinated against yellow fever and MMR simultaneously or at intervals of 30 days or more between doses, had shown low seroconversion for mumps regardless of the interval between administration of the two vaccines. The current study showed 89.5% (95% confidence interval: 83.3; 94.0) seroconversion rate for mumps. All children seroconverted for measles and rubella. After revaccination, high antibody titres and seroconversion rates were achieved against mumps. The results of this study and others suggest that two MMR doses confer optimal immunoresponses for all three antigens and the possible need for additional doses should be studied taking into account not only serological, but also epidemiological data, as there is no serological correlate of protection for mumps.

Clinical features and laboratory findings of human parvovirus B19 in human immunodeficiency virus-infected patients

Immunocompromised patients may develop severe chronic anaemia when infected by human parvovirus B19 (B19V). However, this is not the case in human immunodeficiency virus (HIV)-infected patients with good adherence to highly active antiretroviral treatment (HAART). In this study, we investigated the clinical evolution of five HIV-infected patients receiving HAART who had B19V infections confirmed by serum polymerase chain reaction. Four of the patients were infected with genotype 1a strains and the remaining patient was infected with a genotype 3b strain. Anaemia was detected in three of the patients, but all patients recovered without requiring immunoglobulin and/or blood transfusions. In all cases, the attending physicians did not suspect the B19V infections. There was no apparent relationship between the infecting genotype and the clinical course. In the HAART era, B19V infections in HIV-positive patients may be limited, subtle or unapparent.