Chitosan membranes modified by contact with poly(acrylic acid).

In this work chitosan membranes modified by contact with poly(acrylic acid) (PAA) aqueous solution at two different temperatures (25 degrees C and 60 degrees C) were obtained. The pure chitosan (CS) membranes, as well as those treated with PAA (CSPAA_25 and CSPAA_60) were characterized by FTIR-ATR, water sorption capacity, thermal analysis (TG/DTG), and scanning electron microscopy (SEM). In addition, in vitro permeation experiments were carried out using metronidazol and sodium sulfamerazine aqueous solutions at 0.1% and 0.2% as model drugs. FTIR-ATR results showed the presence of absorption bands of NH3+ and COO(-) indicating the formation of a polyelectrolyte complex between chitosan and poly(acrylic acid). The results also indicated that PAA penetrates deeper into the membrane at higher temperature (60 degrees C), forming a thicker complex layer. Polyelectrolyte complex formation as well as the influence of treatment temperature was confirmed by lower hydrophilicity, higher thermal stability, and lower permeability of the treated membranes. The results show that the methodology used is a simple and very efficient way to drastically change some membrane properties, especially their permeability.

Wild dengue virus types 1, 2 and 3 viremia in rhesus monkeys.

Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.

Wild dengue virus types 1, 2 and 3 viremia in rhesus monkeys

Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.

Adsorption of phenol on formaldehyde-pretreated Pinus pinaster bark: equilibrium and kinetics.

This work studies phenol adsorption on Pinus pinaster bark that has been previously treated with formaldehyde in acid medium. The influence of several variables such as solid/liquid ratio, pH and initial concentration of phenol in the solution on the adsorption capacity of the bark has been analysed. A kinetic model based on phenol diffusion within the pores of the adsorbent was in agreement with the results obtained for high initial concentrations of phenol, allowing the determination of diffusion coefficients. Adsorption equilibrium data were fitted by the Freundlich and BET isotherms. From their parameters phenol adsorption capacity and intensity, as well as the specific surface (BET) of the adsorbent, were determined.

DNA vaccines against dengue virus based on the ns1 gene: the influence of different signal sequences on the protein expression and its correlation to the immune response elicited in mice.

We analyzed four DNA vaccines based on DENV-2 NS1: pcENS1, encoding the C-terminal from E protein plus the NS1 region; pcENS1ANC, similar to pcENS1 plus the N-terminal sequence from NS2a (ANC); pcTPANS1, coding the t-PA signal sequence fused to NS1; and pcTPANS1ANC, similar to pcTPANS1 plus the ANC sequence. The NS1 was detected in lysates and culture supernatants from pcTPANS1-, pcENS1- and pcENS1ANC-transfected cells and not in cells with pcTPANS1ANC. Only the pcENS1ANC leads the expression of NS1 in plasma membrane, confirming the importance of ANC sequence for targeting NS1 to cell surface. High levels of antibodies recognizing conformational epitopes of NS1 were induced in mice immunized with pcTPANS1 and pcENS1, while only few pcENS1ANC-inoculated animals presented detectable anti-NS1 IgG. Protection against DENV-2 was verified in pcTPANS1- and pcENS1-immunized mice, although the plasmid pcTPANS1 induced slight higher protective immunity. These plasmids seem to activate distinct patterns of the immune system.

Construction and biological properties of yellow fever 17D/dengue type 1 recombinant virus.

Dengue virus, a mosquito-borne flavivirus, is one of the most formidable public health threats in tropical and subtropical regions. As yet, there is no licensed vaccine to protect against the disease. A chimeric yellow fever (YF) 17D/dengue (DEN) type 1 virus was constructed by replacing the pre-membrane and envelope genes of YF 17D virus with those from DEN 1 VeMir95 virus, a Venezuelan isolate. The chimeric YF 17D/DEN 1 VeMir95 virus was regenerated from full-length infectious clones stably propagated in Escherichia coli by transfection of Vero cells with in vitro transcribed RNA. The chimeric virus proliferated efficiently in Vero cells ( approximately 6.6 log(10) plaque-forming units/ml). The chimeric virus was not neurovirulent to 3-week-old Swiss Webster mice inoculated by the intracerebral route, in contrast to the YF 17DD vaccine strain that was lethal for 90% of the mice. The YF 17D/DEN 1 virus at Passage 6 was more attenuated for rhesus monkeys than the YF 17DD commercial vaccine after intracerebral inoculation according to the standard neurovirulence test. This virus is a potential candidate to be included in a tetravalent DEN vaccine formulation. The availability of the cloned cDNA allows further structure/function studies on the viral envelope.

DNA vaccine against the non-structural 1 protein (NS1) of dengue 2 virus.

Dengue is one of the most important mosquito-borne viral disease causing dengue fever and/or dengue shock syndrome/haemorrhagic fever. In some reports, the non-structural protein 1 (NS1) has been identified as a promising antigen for the development of vaccines against dengue virus (DENV). Apparently, it can elicit a protective antibody response with complement-fixing activities. In order to investigate the potential of a DNA vaccine based on the NS1 protein against DENV, we used the plasmid pcTPANS1, which contains the secretory signal sequence derived from human tissue plasminogen activator (t-PA) fused to the full length of the DENV-2 NS1 gene. All Balb/c mice intramuscularly inoculated with the pcTPANS1 presented high levels of NS1-specifc antibodies. Vaccinated animals were challenged with intracerebral DENV-2 virus inoculations and a 100% survival was observed. In general, results demonstrate that the pcTPANS1 plasmid is able to induce protection in mice, and then may be used as a vaccination approach against DENV in further assays.

Attenuation and immunogenicity of recombinant yellow fever 17D-dengue type 2 virus for rhesus monkeys

A chimeric yellow fever (YF)-dengue serotype 2 (dengue 2) virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus. Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.

Attenuation and immunogenicity of recombinant yellow fever 17D-dengue type 2 virus for rhesus monkeys.

A chimeric yellow fever (YF)-dengue serotype 2 (dengue 2) virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus. Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.

Comparative sequence analysis of the P-, M- and L-coding region of the measles virus CAM-70 live attenuated vaccine strain

Measles virus is a highly contagious agent which causes a major health problem in developing countries. The viral genomic RNA is single-stranded, nonsegmented and of negative polarity. Many live attenuated vaccines for measles virus have been developed using either the prototype Edmonston strain or other locally isolated measles strains. Despite the diverse geographic origins of the vaccine viruses and the different attenuation methods used, there was remarkable sequence similarity of H, F and N genes among all vaccine strains. CAM-70 is a Japanese measles attenuated vaccine strain widely used in Brazilian children and produced by Bio-Manguinhos since 1982. Previous studies have characterized this vaccine biologically and genomically. Nevertheless, only the F, H and N genes have been sequenced. In the present study we have sequenced the remaining P, M and L genes (approximately 1.6, 1.4 and 6.5 kb, respectively) to complete the genomic characterization of CAM-70 and to assess the extent of genetic relationship between CAM-70 and other current vaccines. These genes were amplified using long-range or standard RT-PCR techniques, and the cDNA was cloned and automatically sequenced using the dideoxy chain-termination method. The sequence analysis comparing previously sequenced genotype A strains with the CAM-70 Bio-Manguinhos strain showed a low divergence among them. However, the CAM-70 strains (CAM-70 Bio-Manguinhos and a recently sequenced CAM-70 submaster seed strain) were assigned to a specific group by phylogenetic analysis using the neighbor-joining method. Information about our product at the genomic level is important for monitoring vaccination campaigns and for future studies of measles virus attenuation.

Comparative sequence analysis of the P-, M- and L-coding region of the measles virus CAM-70 live attenuated vaccine strain.

Measles virus is a highly contagious agent which causes a major health problem in developing countries. The viral genomic RNA is single-stranded, nonsegmented and of negative polarity. Many live attenuated vaccines for measles virus have been developed using either the prototype Edmonston strain or other locally isolated measles strains. Despite the diverse geographic origins of the vaccine viruses and the different attenuation methods used, there was remarkable sequence similarity of H, F and N genes among all vaccine strains. CAM-70 is a Japanese measles attenuated vaccine strain widely used in Brazilian children and produced by Bio-Manguinhos since 1982. Previous studies have characterized this vaccine biologically and genomically. Nevertheless, only the F, H and N genes have been sequenced. In the present study we have sequenced the remaining P, M and L genes (approximately 1.6, 1.4 and 6.5 kb, respectively) to complete the genomic characterization of CAM-70 and to assess the extent of genetic relationship between CAM-70 and other current vaccines. These genes were amplified using long-range or standard RT-PCR techniques, and the cDNA was cloned and automatically sequenced using the dideoxy chain-termination method. The sequence analysis comparing previously sequenced genotype A strains with the CAM-70 Bio-Manguinhos strain showed a low divergence among them. However, the CAM-70 strains (CAM-70 Bio-Manguinhos and a recently sequenced CAM-70 submaster seed strain) were assigned to a specific group by phylogenetic analysis using the neighbor-joining method. Information about our product at the genomic level is important for monitoring vaccination campaigns and for future studies of measles virus attenuation.

Construction, characterization and immunogenicity of recombinant yellow fever 17D-dengue type 2 viruses.

Chimeric yellow fever (YF)-dengue type 2 (Den 2) viruses were constructed by replacing the premembrane (prM) and envelope (E) genes of YF 17D virus with those from Den 2 virus strains of south-east Asian genotype. Whereas viable chimeric viruses were successfully recovered when the YF 17D C gene and the Den 2 prM gene were fused at the signalase cleavage site, no virus could be rescued from the constructions fused at the viral protease cleavage site. Unlike YF virus that replicated in all the cell lines tested and similar to the Den 2 virus, the recombinant viruses did not replicate in vaccine-production certified CEF and MRC5 cells. Besides, chimeric 17D/Den 2 viruses and their parental viruses reached similar growth titers in Vero and C6/36 cell cultures. Analysis of mouse neurovirulence, performed by intracerebral inoculation, demonstrated that the 17D/Den 2 chimera is more attenuated in this system than the YF 17DD virus. Immunization of mice with this chimera induced a neutralizing antibody response associated with a partial protection against an otherwise lethal dose of mouse neurovirulent Den 2 NGC virus. Overall, these results provide further support for the use of chimeric viruses as an attractive methodology for the development of new live flavivirus vaccines.

Phenotypic and molecular analyses of yellow fever 17DD vaccine viruses associated with serious adverse events in Brazil.

The yellow fever (YF) 17D virus is one of the most successful vaccines developed to data. Its use has been estimated to be over 400 million doses with an excellent record of safety. In the past 3 years, yellow fever vaccination was intensified in Brazil in response to higher risk of urban outbreaks of the disease. Two fatal adverse events temporally associated with YF vaccination were reported. Both cases had features similar to yellow fever disease, including hepatitis and multiorgan failure. Two different lots of YF 17DD virus vaccine were administered to the affected patients and also to hundreds of thousands of other individuals without any other reported serious adverse events. The lots were prepared from the secondary seed, which has been in continuous use since 1984. Nucleotide sequencing revealed minor variations at some nucleotide positions between the secondary seed lot virus and the virus isolates from patients; these differences were not consistent across the isolates, represented differences in the relative amount of each nucleotide in a heterogeneous position, and did not result in amino acid substitutions. Inoculation of rhesus monkeys with the viruses isolated from the two patients by the intracerebral (ic) or intrahepatic (ih) route caused minimal viremia and no clinical signs of infection or alterations in laboratory markers. Central nervous system histological scores of rhesus monkeys inoculated ic were within the expected range, and there were no histopathological lesions in animals inoculated ih. Altogether, these results demonstrated the genetic stability and attenuated phenotype of the viruses that caused fatal illness in the two patients. Therefore, the fatal adverse events experienced by the vaccinees are related to individual, genetically determined host factors that regulate cellular susceptibility to yellow fever virus. Such increased susceptibility, resulting in clinically overt disease expression, appears to be extremely rare.

[Seroconversion study of Biken CAM-70 measles vaccine formulations]. / Estudo de soroconversão com formulações da vacina Biken CAM-70 contra sarampo.

OBJECTIVES: To compare seroconversion rates induced by Biken CAM-70 measles vaccines at different viral concentrations. METHODS: Healthy children aged 9 to 18 months from a primary health care unit in Rio de Janeiro, Brazil, and whose guardians agreed with their participation, were randomly assigned to receive one of the following vaccine formulations: 5,000, 1,000 or 200 CCID50 (50% Tissue Culture Infective Dose). The research team, participants, and data analysts were blinded to the type of vaccine administered. Pre- and post-vaccination antibody levels were assessed through Plaque Reduction Neutralization Test. Two interim data analyses were planned to assess unequivocal evidence of the superiority of one of the vaccine types. RESULTS: From 223 recruited children, 84% completed the whole course. Of them, 79% were less than 10 months of age, and 93% did not show detectable measles antibodies in pre-vaccination serum. Seroconversion (four-fold increase in antibody levels) in groups vaccinated with 5,000, 1,000 or 200 CCID50, were 82%, 55%, and 37% (p<0.0000), respectively. Differences in the mean concentration of post-vaccination antibodies were also substantial and statistically significant (p<0.000). Seroconversion rates (pooling data from all vaccine formulations) were 73% to children aged 10 months or more, and 53% in those below 10 months. CONCLUSIONS: Vaccines with concentrations below 5,000 CCID50 did not produce satisfactory seroconversion rates. The vaccine performance by age was consistent with that seen in other studies using Biken CAM-70 strain in which a sizable proportion of 9-month-old children failed to achieve full immunological response.

The yellow fever 17D vaccine virus as a vector for the expression of foreign proteins: development of new live flavivirus vaccines.

The Flaviviridae is a family of about 70 mostly arthropod-borne viruses many of which are major public health problems with members being present in most continents. Among the most important are yellow fever (YF), dengue with its four serotypes and Japanese encephalitis virus. A live attenuated virus is used as a cost effective, safe and efficacious vaccine against YF but no other live flavivirus vaccines have been licensed. The rise of recombinant DNA technology and its application to study flavivirus genome structure and expression has opened new possibilities for flavivirus vaccine development. One new approach is the use of cDNAs encopassing the whole viral genome to generate infectious RNA after in vitro transcription. This methodology allows the genetic mapping of specific viral functions and the design of viral mutants with considerable potential as new live attenuated viruses. The use of infectious cDNA as a carrier for heterologous antigens is gaining importance as chimeric viruses are shown to be viable, immunogenic and less virulent as compared to the parental viruses. The use of DNA to overcome mutation rates intrinsic of RNA virus populations in conjunction with vaccine production in cell culture should improve the reliability and lower the cost for production of live attenuated vaccines. The YF virus despite a long period ignored by researchers probably due to the effectiveness of the vaccine has made a come back, both in nature as human populations grow and reach endemic areas as well as in the laboratory being a suitable model to understand the biology of flaviviruses in general and providing new alternatives for vaccine development through the use of the 17D vaccine strain.

Recent immunization against measles does not interfere with the sero-response to yellow fever vaccine.

In order to determine whether previous measles vaccination interferes with the sero-response to yellow fever vaccine, 294 children at nine months of age were randomly assigned to immunization with yellow fever vaccine at different time intervals after measles vaccination. The seroconversion rate (SCR) and the log10 geometric mean titer (GMT) for 17 DD yellow fever vaccine at different intervals after Schwarz measles vaccination were: 1-6 days: SCR = 44/57 = 77%; GMT = 4.57; 7-13 days: SCR = 36/53 = 68%; GMT = 4.46; 14-21 days: SCR = 55/65 = 85%; GMT = 4.46; 22-27 days: SCR = 41/54 = 76%; GMT = 4.41 and >28 days: SCR = 52/65 = 80%; GMT = 4.24 (p > 0.05). We conclude that recent immunization against measles does not interfere with the sero-response to yellow fever vaccine.

The yellow fever 17D vaccine virus: molecular basis of viral attenuation and its use as an expression vector.

The yellow fever (YF) virus is the prototype flavivirus. The use of molecular techniques has unraveled the basic mechanisms of viral genome structure and expression. Recent trends in flavivirus research include the use of infectious clone technology with which it is possible to recover virus from cloned cDNA. Using this technique, mutations can be introduced at any point of the viral genome and their resulting effect on virus phenotype can be assessed. This approach has opened new possibilities to study several biological viral features with special emphasis on the issue of virulence/attenuation of the YF virus. The feasibility of using YF virus 17D vaccine strain, for which infectious cDNA is available, as a vector for the expression of heterologous antigens is reviewed.

The yellow fever 17D vaccine virus: molecular basis of viral attennuation and it use as an expression vector

The yellow fever (YF) virus is the prototype flavivirus. The use of molecular techniques has unraveled the basic mechanisms of viral genome structure and expression. Recent trends in flavivirus research include the use of infectious clone technology with which it is possible to recover virus from cloned cDNA. Using this technique, mutations can be introduced at any point of the viral genome and their resulting effect on virus phenotype can be assessed. This approach has opened new possibilities to study several biological viral features with special emphasis on the issue of virulence/attenuation of the YF virus. The feasibility of using YF virus 17D vaccine strain, for which infectious cDNA is available, as a vector for the expression of heterologous antigens is reviewed.