Luciane P. Gaspar

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.

Postoperative irradiation of primary or recurrent pterygium: results and sequelae.

PURPOSE To evaluate the efficacy of postoperative beta irradiation and to analyze treatment sequelae in patients with primary and recurrent pterygium. METHODS AND MATERIALS From June 1986 to June 1998, 94 patients corresponding to 100 eyes received postoperative beta irradiation. Two groups of patients were treated: 37 eyes with primary pterygium (Group I) and 63 eyes with recurrent pterygium (Group II). Terson technique surgery was used in the majority of patients. Time between surgery and beta irradiation ranged from 2 to 48 h. Radiation doses and fractionation consisted of 30 Gy/3 fractions/5 days in 17 cases, 60 Gy/6 fractions/6 weeks in 80 cases, and 20 Gy/1 fraction in 3 patients. RESULTS Fourteen of the 100 cases (14%) treated with surgery and adjuvant irradiation recurred. The overall crude local recurrence rates were 5.4% for Group I and 19% for Group II patients. The 5-year probability of local tumor control was 83.5% for the whole group of patients, 94% for Group I, and 76.9% for Group II (p = 0.04). The early sequelae related to surgery or irradiation were self limited and disappeared by 6 months after the end of the treatment: ocular irritation (14 cases), scleral atrophy (5 cases), and neovascularization (7 cases). A greater incidence of sequelae was observed in Group II patients, but the difference between the groups was not statistically significant (p = 0.15). No significant correlation between treatment sequelae and treatment dose was noted: 29% sequelae with 30 Gy vs. 18.7% sequelae with 60 Gy (p = 0.32). No late complications have been observed. CONCLUSION Adjuvant beta irradiation provides effective therapy for primary pterygium, is somewhate less effective in patients with recurrent pterygium, and is associated with a moderate rate of early and transient sequelae.

Declining prevalence of hepatitis A virus antibodies among children from low socioeconomic groups reinforces the need for the implementation of hepatitis A vaccination in Brazil

Age-related seroprevalence studies that have been conducted in Brazil have indicated a transition from a high to a medium endemicity of hepatitis A virus (HAV) infection in the population. However, most of these studies have focused on urban populations that experience lower incidence rates of HAV infection. In the current study, the prevalence of anti-HAV antibodies was investigated in children with a low socioeconomic status (SES) that live on the periphery of three capital cities in Brazil. A total of 1,162 dried blood spot samples were collected from individuals whose ages ranged from one-18 years and tested for anti-HAV antibodies. A large number of children under five years old (74.1-90%) were identified to be susceptible to HAV infection. The anti-HAV antibody prevalence reached ≥ 50% among those that were 10-14 years of age or older. The anti-HAV prevalence rates observed were characteristics of regions with intermediate level of hepatitis A endemicity. These data indicated that a large proportion of children with a low SES that live at the periphery of urban cities might be at risk of contracting an HAV infection. The hepatitis A vaccine that is currently offered in Brazil is only available for high-risk groups or at private clinics and is unaffordable for individuals with a lower SES. The results from this study suggest that the hepatitis A vaccine should be included in the Brazilian National Program for Immunisation.

Production of yellow fever virus in microcarrier-based Vero cell cultures

In this work, the propagation of the 17DD yellow fever virus in Vero cells grown on Cytodex-1 microcarriers was evaluated. After verifying that upon infection the virus adsorption step could be performed under continuous agitation, experiments were carried out in spinners and sparged lab-scale stirred-tank bioreactor to evaluate the use of a commercial serum-free medium (VP-SFM) and to investigate the effects of multiplicity of infection (MOI) and time of infection (TOI) on virus production. Virus titers as high as 8.4 x 10(8)pfu/mL were obtained upon infection with MOI of 0.02 and TOI of 3 days, using the serum-free medium in the sparged bioreactor.

The Use of Dried Blood Spots for Assessing Antibody Response to Hepatitis A Virus After Natural Infection and Vaccination

During recent years, vaccination against hepatitis A has been implemented in several countries. It is expected that the increase in mass vaccination against hepatitis A will eventually result in a decreased prevalence of anti‐HAV antibodies in the general population. For this reason, a suitable clinical sample for diagnosis of hepatitis A must be sufficiently sensitive to enable detection of lower antibodies titers. In this study, the feasibility of using dried blood spots (DBS) was assessed for the detection of anti‐HAV antibodies after a natural infection and vaccination. Seventy‐four DBS and paired plasma samples were obtained from a group of college students for a cross‐sectional hepatitis A seroepidemiological study. Forty‐six students seronegative for anti‐HAV were selected randomly and immunized with an inactivated hepatitis A vaccine using an 0–6 month schedule. Seroconversion was monitored in paired plasma and DBS samples 6 months after the first dose followed by a period of 8 and 24 months after the second dose. A strong correlation between OD/CO rates of paired plasma and DBS samples for the detection of anti‐HAV was observed. The sensitivity and specificity of the DBS compared with plasma for the detection of anti‐HAV antibodies after natural infection was 100%. The sensitivity of DBS in samples collected 24 months after the second dose of hepatitis A vaccine was 95.4%. The results showed that DBS samples can be used for the detection of anti‐HAV antibodies both after natural infection or vaccination. J. Med. Virol. 83:208–217, 2011.

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 β-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.

Effects of hydrostatic pressure on the stability and thermostability of poliovirus: A new method for vaccine preservation

Viruses are a structurally diverse group of infectious agents that differ widely in their sensitivities to high hydrostatic pressure (HHP). Studies on picornaviruses have demonstrated that these viruses are extremely resistant to HHP treatments, with poliovirus appearing to be the most resistant. Here, the three attenuated poliovirus serotypes were compared with regard to pressure and thermal resistance. We found that HHP does not inactivate any of the three serotypes studied (1-3). Rather, HHP treatment was found to stabilize poliovirus by increasing viral thermal resistance at 37 degrees C. Identification of new methods that stabilize poliovirus against heat inactivation would aid in the design of a more heat-stable vaccine, circumventing the problems associated with refrigeration during storage and transport of the vaccine prior to use.

Development of a membrane adsorber based capture step for the purification of yellow fever virus.

Yellow fever (YF) is an endemic disease in some tropical areas of South America and Africa that presents lethality rate between 20 and 50%. There is no specific treatment and to control this disease a highly effective live-attenuated egg based vaccine is widely used for travelers and residents of areas where YF is endemic. However, recent reports of rare, sometimes fatal, adverse events post-vaccination have raised concerns. In order to increase safety records, alternative strategies should be considered, such as developing a new inactivated vaccine using a cell culture based technology, capable of meeting the demands in cases of epidemic. With this goal, the production of YF virus in Vero cells grown on microcarriers and its subsequent purification by chromatographic techniques was studied. In this work we investigate the capture step of the purification process of the YF virus. At first, virus stability was studied over a wide pH range, showing best results for the alkaline region. Considering this result and the pI of the envelope protein previously determined in silico, a strong anion exchanger was considered most suitable. Due to the easy scalability, simplicity to handle, absence of diffusional limitations and suitability for virus handling of membrane adsorbers, a Q membrane was evaluated. The amount of antigen adsorbed onto the membrane was investigated within the pH range for virus stability, and the best pH for virus adsorption was considered to be 8.5. Finally, studies on gradient and step elution allowed to determine the most adequate salt concentration for washing (0.15M) and virus elution (0.30 M). Under these operating conditions, it was shown that this capture step is quite efficient, showing high product recovery (93.2±30.3%) and efficient DNA clearance (0.9±0.3 ng/dose).

Increasing Vero viable cell densities for yellow fever virus production in stirred-tank bioreactors using serum-free medium.

In this work, changes in Vero cell cultivation methods have been employed in order to improve cell growth conditions to obtain higher viable cell densities and to increase viral titers. The propagation of the 17DD yellow fever virus (YFV) in Vero cells grown on Cytodex I microcarriers was evaluated in 3-L bioreactor vessels. Prior to the current changes, Vero cells were repeatedly displaying insufficient microcarrier colonization. A modified cultivation process with four changes has resulted in higher cell densities and higher virus titers than previously observed for 17DD YFV.