New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters.

The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.

An inactivated yellow fever 17DD vaccine cultivated in Vero cell cultures.

Yellow fever is an acute infectious disease caused by prototype virus of the genus Flavivirus. It is endemic in Africa and South America where it represents a serious public health problem causing epidemics of hemorrhagic fever with mortality rates ranging from 20% to 50%. There is no available antiviral therapy and vaccination is the primary method of disease control. Although the attenuated vaccines for yellow fever show safety and efficacy it became necessary to develop a new yellow fever vaccine due to the occurrence of rare serious adverse events, which include visceral and neurotropic diseases. The new inactivated vaccine should be safer and effective as the existing attenuated one. In the present study, the immunogenicity of an inactivated 17DD vaccine in C57BL/6 mice was evaluated. The yellow fever virus was produced by cultivation of Vero cells in bioreactors, inactivated with ß-propiolactone, and adsorbed to aluminum hydroxide (alum). Mice were inoculated with inactivated 17DD vaccine containing alum adjuvant and followed by intracerebral challenge with 17DD virus. The results showed that animals receiving 3 doses of the inactivated vaccine (2 µg/dose) with alum adjuvant had neutralizing antibody titers above the cut-off of PRNT50 (Plaque Reduction Neutralization Test). In addition, animals immunized with inactivated vaccine showed survival rate of 100% after the challenge as well as animals immunized with commercial attenuated 17DD vaccine.

Evaluation of accuracy and reliability of the plaque reduction neutralization test (micro-PRNT) in detection of yellow fever virus antibodies.

Yellow fever is a disease caused by the prototype virus of the genus Flavivirus and remains endemic in tropical forest regions from Africa and South America, despite the availability of effective vaccines. These are capable of inducing a rapid specific immune response, with the formation of neutralizing antibodies that appear early, are protective and long lasting. The Plaque Reduction Neutralization Test is considered the most sensitive and specific test for quantification of neutralizing antibodies, and the reference method for assessing the protective immune response after vaccination. This study evaluated the reliability (repeatability and reproducibility) and accuracy (sensitivity, specificity and overall accuracy) of micro-PRNT50 and compared its performance with the micro-PRNT90. Although the micro-PRNT50 has showed satisfactory levels of reliability (ICCs ranged from 0.62 to 0.NorNormas e Manuais Técnicosas e Manuais Técnicos6 for repeatability and 0.72 for reproducibility) and accuracy (sensitivity of 91.1%, specificity of 72.9% and overall accuracy of 78%), the micro-PRNT90 showed higher performance, with ICCs for repeatability ranged from 0.78 to 0.79 and 0.81 for reproducibility, sensitivity of 100%, specificity of 94.7% and overall accuracy of 95%. Modifications in the test methodology and changes in the classification criteria in the readings of the results obtained will be important to improve the accuracy of micro-PRNT.

DNA vaccines against dengue virus type 2 based on truncate envelope protein or its domain III.

Two DNA vaccines were constructed encoding the ectodomain (domains I, II and III) of the DENV2 envelope protein (pE1D2) or only its domain III (pE2D2), fused to the human tissue plasminogen activator signal peptide (t-PA). The expression and secretion of recombinant proteins was confirmed in vitro in BHK cells transfected with the two plasmids, detected by immunofluorescence or immunoprecipitation of metabolically labeled gene products, using polyclonal and monoclonal antibodies against DENV2. Besides, results reveal that the ectodomain of the E protein can be efficiently expressed in vivo, in a mammalian system, without the prM protein that is hypothesized to act as a chaperonin during dengue infection. Balb/c mice were immunized with the DNA vaccines and challenged with a lethal dose of DENV2. All pE1D2-vaccinated mice survived challenge, while 45% of animals immunized with the pE2D2 died after infection. Furthermore, only 10% of pE1D2-immunized mice presented some clinical signs of infection after challenge, whereas most of animals inoculated with the pE2D2 showed effects of the disease with high morbidity degrees. Levels of neutralizing antibodies were significantly higher in pE1D2-vaccinated mice than in pE2D2-immunized animals, also suggesting that the pE1D2 vaccine was more protective than the pE2D2.

Risk factors for dengue virus infection in rural Amazonia: population-based cross-sectional surveys.

A comparison of dengue virus (DENV) antibody levels in paired serum samples collected from predominantly DENV-naive residents in an agricultural settlement in Brazilian Amazonia (baseline seroprevalence, 18.3%) showed a seroconversion rate of 3.67 episodes/100 person-years at risk during 12 months of follow-up. Multivariate analysis identified male sex, poverty, and migration from extra-Amazonian states as significant predictors of baseline DENV seropositivity, whereas male sex, a history of clinical diagnosis of dengue fever, and travel to an urban area predicted subsequent seroconversion. The laboratory surveillance of acute febrile illnesses implemented at the study site and in a nearby town between 2004 and 2006 confirmed 11 DENV infections among 102 episodes studied with DENV IgM detection, reverse transcriptase-polymerase chain reaction, and virus isolation; DENV-3 was isolated. Because DENV exposure is associated with migration or travel, personal protection measures when visiting high-risk urban areas may reduce the incidence of DENV infection in this rural population.

Pressure-inactivated yellow fever 17DD virus: implications for vaccine development.

The successful Yellow Fever (YF) vaccine consists of the live attenuated 17D-204 or 17DD viruses. Despite its excellent record of efficacy and safety, serious adverse events have been recorded and influenced extensive vaccination in endemic areas. Therefore, alternative strategies should be considered, which may include inactivated whole virus. High hydrostatic pressure has been described as a method for viral inactivation and vaccine development. The present study evaluated whether high hydrostatic pressure would inactivate the YF 17DD virus. YF 17DD virus was grown in Vero cells in roller bottle cultures and subjected to 310MPa for 3h at 4 degrees C. This treatment abolished YF infectivity and eliminated the ability of the virus to cause disease in mice. Pressure-inactivated virus elicited low level of neutralizing antibody titers although exhibited complete protection against an otherwise lethal challenge with 17DD virus in the murine model. The data warrant further development of pressure-inactivated vaccine against YF.

Attenuation of recombinant yellow fever 17D viruses expressing foreign protein epitopes at the surface.

The yellow fever (YF) 17D vaccine is a live attenuated virus. Three-dimensional (3D) homology modeling of the E protein structure from YF 17D virus and its comparison with that from tick-borne encephalitis virus revealed that it is possible to accommodate inserts of different sizes and amino acid compositions in the flavivirus E protein fg loop. This is consistent with the 3D structures of both the dimeric and trimeric forms in which the fg loop lies exposed to solvents. We demonstrate here that YF 17D viruses bearing foreign humoral (17D/8) and T-cell (17D/13) epitopes, which vary in sequence and length, displayed growth restriction. It is hypothesized that interference with the dimer-trimer transition and with the formation of a ring of such trimers in order to allow fusion compromises the capability of the E protein to induce fusion of viral and endosomal membranes, and a slower rate of fusion may delay the extent of virus production. This would account for the lower levels of replication in cultured cells and of viremia in monkeys, as well as for the more attenuated phenotype of the recombinant viruses in monkeys. Testing of both recombinant viruses (17D/8 and 17D/13) for monkey neurovirulence also suggests that insertion at the 17D E protein fg loop does not compromise the attenuated phenotype of YF 17D virus, further confirming the potential use of this site for the development of new live attenuated 17D virus-based vaccines.

Production of yellow fever 17DD vaccine virus in primary culture of chicken embryo fibroblasts: yields, thermo and genetic stability, attenuation and immunogenicity.

While a good vaccine against yellow fever (YF) virus has been available for decades, the basic technology for the production of YF vaccine in chicken embryos has remained substantially unchanged since the 1940s. Here we describe the highly efficient and economic production of the 17DD strain of YF virus in chicken embryo fibroblast (CEF) cell cultures with viral titers ranging from 6.3 to 6.7 log10PFU/mL. Thermostability of two different formulations (5 and 50-dose vials) of the CEF vaccine virus was found to be as high as the current vaccines retaining the minimal titer required for YF 17D vaccines. The production passage in CEF did not lead to the selection of genetic variants as shown by nucleotide sequence analyses of the CEF-derived vaccine lots or the sequence of viruses recovered from monkeys experimentally inoculated with the CEF virus. YF 17DD virus produced in CEF was also indistinguishable from its seed lot virus parent in terms of plaque size and immunogenicity in mice and monkeys. Comparison of the CEF virus and the seed lot virus made in chicken embryo in the internationally accepted monkey neurovirulence test (MNVT) revealed a higher clinical score for the former. The differences in central nervous system (CNS) histological scores for monkeys inoculated with the chicken embryo and experimental CEF vaccines were at the borderline level of statistical significance. These data warrant further studies on the monkey attenuation of other batches of CEF-derived vaccines.

Estudo de soroconversäo com formulaçöes da vacina Biken CAM-70 contra sarampo / Comparison of seroconversion rates with CAM-70 measles vaccine at different dosages

Objetivo: Comparar a resposta sorológica induzida por formulaçöes com diferentes concentraçöes de vírus da vacina contra sarampo da cepa Biken CAM-70. Métodos: Crianças sadias de 9 a 18 meses de um centro de saúde do Rio de Janeiro, RJ, cujos responsáveis concordaram em participar, foram randomizadas em três grupos vacinados com concentraçöes de 5.000, 1.000 ou 200 CCID50 (50 por cento Tissue Culture Infective Dose). Os participantes e o pessoal da pesquisa ignoravam o tipo de vacina administrada. A avaliaçäo sorológica foi realizada pelo teste de reduçäo em plaque de lise. Duas análises intermediárias dos dados foram programadas. Resultados: Das 223 crianças recrutadas, 84 por cento completaram todos os procedimentos; 79 por cento tinham idade menor que 10 meses; 93 por cento näo tinham anticorpos contra sarampo no soro pré-vacinal. As proporçöes de soroconversäo (quadruplicaçäo das concentraçöes pré-vacinais) foram 82 por cento, 55 por cento e 37 por cento (p<0,0000), nos grupos vacinados com 5.000, 1.000 ou 200 CCID50, respectivamente. As diferenças nas concentraçöes médias de anticorpos pós-vacinais também foram substanciais e estatisticamente significativas (p<0,000). A soroconversäo (independente da formulaçäo da vacina) foi de 73 por cento nas crianças com 10 ou mais meses de idade e 53 por cento naquelas com menos de 10 meses. Conclusöes: Formulaçöes da vacina com concentraçöes inferiores a 5.000 CCID50 näo induziram soroconversäo satisfatória. O desempenho da vacina por faixa etária foi compatível com o observado em outros estudos com a vacina Biken CAM-70 e indica que uma proporçäo apreciável de crianças vacinadas aos 9 meses pode näo obter resposta imunológica plena