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We describe the validation of an enzyme-linked immunosorbent assay (ELISA) and confirmatory immunoblotting assays based on a recombinant p30 protein (p30r) produced in insect larvae using a baculovirus vector. Such validation included the following: (i) the scaling up and standardization of p30r production and the associated immunoassays, (ii) a broad immunological analysis using a large number of samples (a total of 672) from Spain and different African locations, and (iii) the detection of the ASF virus (ASFV)-antibody responses at different times after experimental infection. Yields of p30r reached up to 15% of the total protein recovered from the infected larvae at 3 days postinfection. Serological analysis of samples collected in Spain revealed that the p30r-based ELISA presented similar sensitivity to and higher specificity than the conventional Office International des Epizooties-approved ASFV ELISA. Moreover, the p30r ELISA was more sensitive than the conventional ELISA test in detecting ASFV-specific antibodies in experimentally infected animals at early times postinfection. Both the recombinant and conventional ELISAs presented variable rates of sensitivity and specificity with African samples, apparently related to their geographical origin. Comparative analyses performed on the sequences, predicted structures, and antigenicities of p30 proteins from different Spanish and African isolates suggested that variability among isolates might correlate with changes in antigenicity, thus affecting detection by the p30r ELISA. Our estimations indicate that more than 40,000 ELISA determinations and 2,000 confirmatory immunoblotting tests can be performed with the p30r protein obtained from a single infected larva, making this a feasible and inexpensive strategy for production of serological tests with application in developing countries.

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Vaccine antigens against rabbit hemorrhagic disease virus (RHDV) are currently derived from inactivated RHDV obtained from livers of experimentally infected rabbits. Several RHDV-derived recombinant immunogens have been reported. However, their application in vaccines has been restricted due to their high production costs. In this paper, we describe the development of an inexpensive, safe, stable vaccine antigen for RHDV. A baculovirus expressing a recombinant RHDV capsid protein (VP60r) was used to infect Trichoplusia ni insect larvae. It reached an expression efficiency of 12.5% of total soluble protein, i.e. approximately 2 mg of VP60r per larva. Preservation of the antigenicity and immunogenicity of the VP60r was confirmed by immunological and immunization experiments. Lyophilized crude larvae extracts, containing VP60r, were stable, at room temperature, for at least 800 days. In all cases, rabbits immunized with a single dose of VP60r by the intramuscular route were protected against RHDV challenge. Doses used were as low as 2 microg of VP60r in the presence of adjuvant or 100 microg without one. Orally administered VP60r in the absence of an adjuvant gave no protection. The potential costs of an RHDV vaccine made using this technology would be reduced considerably compared with producing the same protein in insect cells maintained by fermentation. In conclusion, the larva expression system may provide a broad-based strategy for production of recombinant subunit antigens (insectigens) for human or animal medicines, especially when production costs restrain their use.