Schema of plasmid DNA constructs used.

All constructs were based on the same mammalian expression vector, regulated with the promoter and enhancer of the early gene of CMV and transcription termination region (polyA) from SV40.16 The genes contained optimized Kozak 5′–untranslated region. The gene for gp120 from HIV-1 89.6 was PCR cloned in frame either with the signal sequence (SL) of murine IP-10 or with chemokine or β-defensin 2 genes (MCP-3, MDC, vMIP2, and mDF2β, respectively). Constructs with gp140-14 consisted of fusion of gp120 with the extracellular domain of gp41 linked via a flexible 14–amino acid peptide (pgp140-14). The gp140-14 was also fused with chemokines, such as human MDC and vMIP2 (pMDCgp140-14 and pvMIP2gp140-14, respectively). All expression cassettes were verified by DNA sequencing, and protein expression was assessed by transient transfection of HEK293 cells.