Increased production capacity is one of the most important priorities for seasonal and pandemic influenza vaccines. In the present study, we used a baculovirus-insect larvae system (considered small, living biofactories) to improve the production of recombinant influenza virus H1N1 hemagglutinin (HA). Insect larvae produced four-fold more HA protein than insect cells per biomass unit (1 g of fresh larvae weight). A single infected Trichoplusia ni larva produced up to 113 mu g of soluble and easily purified recombinant HA, an amount similar to that produced by 1.2 x 10(8) Sf21 insect cells infected by the same baculovirus. The use of the KDEL endoplasmic reticulum retention signal fused to the HA protein further increased recombinant protein production. Larvae-derived HA was immunogenically functional in vaccinated mice, inducing the generation of hemagglutination inhibition antibodies and a protective immune response against a lethal challenge with a highly virulent virus. The productivity, scalability and cost efficiency of small, living biofactories based on insect larvae suggest a broad-based strategy for the production of recombinant subunit vaccines against seasonal or pandemic influenza as an alternative to fermentation technologies. (C) 2011 Elsevier Inc. All rights reserved.