Quantitative ELISA sandwich for a new vaccine against avian influenza virus H5N1.

Analytical techniques are essential in the process of standardizing and validating vaccines. In this study we described a methodology to establish an ELISA sandwich for the quantification of a new vaccine against avian influenza virus H5N1 based on the main antigenic determinant of the virus, the extracellular domain of the glycoprotein hemagglutinin (HA), fused to the extracellular domain of the chicken CD154 glycoprotein (HACD). The chimerical proteins HA and HACD were produced in SiHa cells and the experiments were performed by using three monoclonal antibodies (MAb-HA1, MAb-HA2 and MAb-HA3), alone or conjugated to horseradish peroxidase (HRP-HA1, HRP-HA2 and HRP-HA3). The hemagglutination inhibition assay was carried out with a negative and a positive H5N2 reference serum, together with the antigen H5N1 A/Mallard/Italy/3401/05, all purchased from the "Istituto Zooprofilattico delle Venezie", Italy. After demonstrating the similar recognition pattern between the HA and the HACD proteins, the MAb-HA2 at a concentration of 2,5 µg/mL was selected as the capture antibody and the HRP-HA3 at a dilution of 1/20000 was selected as the detection antibody due to their optimal values of optical density at these conditions. The best dynamic range of the standard curve using the protein HACD was achieved at concentrations from 100 to 1,56 ng/mL. There were no significant differences when five batches of HACD were quantified by the ELISA sandwich and the bicinchoninic acid method linked to densitometry. In conclusion, the final parameters for the quantification of the chimeric protein HACD using an ELISA sandwich were described, which could contribute to develop a large-scale process for the final vaccine production.

Integrated control of Boophilus microplus ticks in Cuba based on vaccination with the anti-tick vaccine Gavac.

Boophilus microplus has developed resistance against a range of chemical acaricides which has stimulated the development of alternative methods such as vaccination against ticks. In Cuba, the Bm86-based recombinant vaccine Gavac has been successfully used in a number of controlled laboratory and field trials in cattle against B. microplus. In this paper, we have evaluated Gavac in a large scale field trial wherein 588,573 dairy cattle were vaccinated with the aim to reduce the number of acaricidal treatments. It was found that the number of acaricidal treatments could be reduced by 87% over a period of 8 years (1995--2003). Prior to the introduction of the vaccine, 54 clinical cases of babesiosis and six fatal cases were reported per 1000 animals. Six years later, the incidence of babesiosis was reduced to 1.9 cases per 1000 cattle and mortality reduced to 0.18 per 1000. The national consumption of acaricides in Cuba could be reduced by 82% after the implementation of the integrated anti-B. microplus control program.