Targeting viral antigens for proteosomal degradation has previously been proposed as a means for immunogenicity augmentation. However, utilization of modified unstable antigens may be insufficient for potent T-cell cross-presentation by APCs, a mechanism that requires high levels of the antigenic protein. Therefore, we hypothesized that a recombinant vaccine utilizing a combination of proteosome-sensitive and proteosome-resistant versions of an antigen in a prime-boost regimen may provide the most efficient CTL response. To address this hypothesis, we utilized conserved proteosome-resistant influenza A virus proteins M1 and NS1. Unstable versions of these polypeptides were constructed by destroying their 3D structure via truncations or short insertions into predicted alpha-helical structures. These modified polypeptides were stabilized in the presence of the proteosome inhibitor MG132, strongly suggesting that they are degraded via a ubiquitin-proteosome pathway. Importantly, with both M1 and NS1antigens, homologous DNA vaccination with a mixture of unstable and proteosome-resistant wt forms of these proteins resulted in significantly higher CTL activity than vaccination with either wt or degradable forms. The most dramatic effect was seen with NS1, where homologous immunization with a mixture of these two forms was the only regimen that produced a notable elevation of CTL response, compared to vaccination with the wt NS1. Additionally, for M1 protein, heterologous vaccination utilizing the unstable form as prime and wild-type form as boost, demonstrated significant augmentation of the CTL response. These data indicate that combining proteosome-sensitive and proteosome-resistant forms of an antigen during vaccination is advantageous. © 2008 Elsevier Ltd. All rights reserved.