Antigenic and physicochemical characterization of Hepatitis B surface protein under extreme temperature and pH conditions.

Hepatitis B virus causes acute and chronic infections in millions of people worldwide and, since 1982, a vaccine with 95% effectiveness has been available for immunization. The main component of the recombinant hepatitis B vaccine is the surface antigen protein (HBsAg). In this work, the effect of pH, ionic strength and temperature on the native state of the HBsAg antigen were studied by a combination of biophysical methods that included small angle X-ray scattering, synchrotron radiation circular dichroism, fluorescence and surface plasmon resonance spectroscopies, as well as in vivo and in vitro potency assays. The native conformation, morphology, radius of gyration, and antigenic properties of the HBsAg antigen demonstrate high stability to pH treatment, especially in the pH range employed in all stages of HBsAg vaccine production and storage. The HBsAg protein presents thermal melting point close to 56 °C, reaching a more unfolded state after crossing this point, but it only experiences loss of vaccine potency and antigenic properties at 100 °C. Interestingly, a 6-month storage period does not affect vaccine stability, and the results are similar when the protein is kept under refrigerated conditions or at room temperature (20 °C). At frozen temperatures, large aggregates (>200 nm) are formed and possibly cause loss of HBsAg content, but that does not affect the in vivo assay. Furthermore, HBsAg has a well-ordered secondary structure content that is not affected when the protein is formulated with silica SBA-15, targeting the oral delivery of the vaccine. The combined results from all the characterization techniques employed in this study showed the high stability of the antigen at different storage temperature and extreme values of pH. These findings are important for considering the delivery of HBsAg to the immune system via an oral vaccine.

[Assessment of the efficacy of antivenom injection at the site of the intramuscular inoculation of Bothrops jararaca venom: experimental study in mice]. / Avaliação da eficácia do antiveneno botrópico administrado no local da inoculação intramuscular do veneno de Bothrops jararaca: estudo experimental em camundongos.

The 50% effective intraperitoneal (ip) dose of Bothrops jararaca antivenom (ED50) was assessed in mice immediately (ED50 Oh) and thirty minutes (ED50 30') after the intramuscular (im) injection of two 50% lethal dose (LD50) of Bothrops jararaca venom. The efficacy of the antivenom injected at the venom inoculation site was assessed by the inoculation of two LD50 of the venom by im route, followed immediately (ED50 Oh) and 30 minutes later (ED50 30') by administration of the ED50 of the antivenom either entirely by the ip route or 50 percent ip plus 50 percent im, at the same inoculation site. It was shown that the ED50 30' was 3 times greater, than the ED50 Oh and that the antivenom was more protective to mice (lower death rate in 48 hours) when given entirely ip. It was concluded that, in this experimental model, a higher dose of bothropic antivenom is needed when the treatment is started lately, and that there is no benefit in its administration at the venom inoculation site.

[Evaluation in mice of the efficacy of antivenins administered at the site of the intramuscular inoculation of the Crotalus durissus terrificus venom]. / Avaliação em camundongo da eficácia do antiveneno administrado no local da inoculação intramuscular do veneno de Crotalus durissus terrificus.

The efficacy of the Crotalus durissus terrificus antivenom administration by intramuscular (im) injection at the same place of the im inoculation, of the C. d. terrificus venom was evaluated in mice. In three experiments two DL50 of the venom were inoculated and the antivenom was administered in three different ways: half of the ED50 by intraperitoneal (ip) administration and half by injection, at the same place, immediately after the venom inoculation and thirty minutes after the im venom inoculation; four fifth of ED50 by ip administration and one fifth by injection, at the same place and thirty minutes after the venom inoculation. The antivenom that was administered by intraperitoneal route provided a higher protection to mice (a lower death rate in a 48 hours period) than when it was administered in parts, by intramuscular injection, at the same place of the venom inoculation (p < 0.05). Therefore, it is concluded that this should not be used in human beings bitten by snakes.