Vaccine 26 (2008) 5170–5176 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine HIV-1 reverse transcriptase artificially targeted for proteasomal degradation induces a mixed Th1/Th2-type immune response Elizaveta S. Starodubova a,b,c , Andreas Boberg b,d , Marina Litvina e , Alexey Morozov a,c , Natalia V. Petrakova c , Andrey Timofeev a , Oleg Latyshev c , Vera Tunitskaya a , Britta Wahren b,d , Maria G. Isaguliants b,c,d,∗ , Vadim L. Karpov a a WA Engelhardt Institute of Molecular Biology, Moscow, Russia b Swedish Institute for Infectious Disease Control, Karolinska Institute, 17182 Stockholm, Sweden c DI Ivanovsky Institute of Virology, 123098 Moscow, Russia d Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, 17182 Stockholm, Sweden e Institute of Immunology, 115478 Moscow, Russia article info Article history: Available online 15 April 2008 Keywords: HIV-1 DNA vaccine Proteasome Reverse transcriptase PEST signal Th1 Th2 Cytokines abstract Targeting of a DNA vaccine encoded protein for degradation via the proteasome is attempted since it may enhance the immunogenicity of the vaccine. We have fused HIV-1 reverse transcriptase (RT) to mouse ornithine decarboxylase (ODC), a protein rapidly degraded by proteasome in an ubiquitine-independent fashion, to enhance the introduction of RT into the MHC class I pathway. We also designed a fusion of RT with two short signals from the C-terminus of ODC (ODCsig) representing a minimal proteasome- targeting moiety of ODC (PEST signal). Fusion to ODC or ODC signal domain led to a marked enhancement of RT degradation. Plasmids encoding RT-ODC and RT-ODCsig chimera were used to immunize BALB/c mice. The administration of the plasmids was not associated with autoimmune disease. Moreover, mice receiving RT-ODCsig gene mounted a mixed Th1/Th2 response characterized by the in vitro secretion of IFN-, IL-2, TNF-, IL-4, and IL-10 upon stimulation of splenocytes with RT protein or RT derived peptides. Serum titers of 10 2 to 10 3 were observed in more than 50% of animals in that group, whereas fewer animals mounted an anti-RT response in the RT-ODC gene immunized group. Chimeras of the type described here can, therefore, be used in vaccinations aiming to induce HIV-1 RT-specific immune response. © 2008 Elsevier Ltd. All rights reserved. 1. Introduction Cytotoxic T lymphocytes are thought to be important in con- trolling HIV replication [1–3]. The induction of a strong Th1-type immune response is thought to be beneficial for a therapeutic vac- cine against HIV [4]. Plasmid DNA encoding foreign proteins have been shown to elicit both humoral and cellular immune response and are therefore of interest for vaccine development [5]. In addi- tion, DNA-vaccine technology provides a tool for manipulating the vaccine component, which may thereby elicit a more potent immune response of Th1- or Th2-type. Targeting the immunogen to proteasomal degradation is one of such approaches thought to enhance the Th1-type immunity. Ornithine decarboxylase (ODC) is rapidly degraded by the proteasome in an ubiquitine-independent ∗ Corresponding author at: Department of Virology, Swedish Institute for Infec- tious Disease Control, Centralf ¨ orr ˚ adet, Tomtebodav¨ agen 12B, 171 82 Solna, Sweden. Tel.: +46 8 4572609; fax: +46 8 337272. E-mail address: maria.isaguliants@smi.ki.se (M.G. Isaguliants). fashion [6,7]. We have previously shown that RT is a stable pro- tein with the half-life about 20 h [10]. Further, we have shown that fusion of RT to ODC leads the RT moieties to an enhanced pro- teasomal degradation making fusions with ODC attractive as gene vaccines [11]. The degradation of ODC is promoted by two signals in the C-terminus of ODC [8,9]. To diminish the risk of develop- ment of autoimmune reaction to ODC component while preserving the proteasomal targeting, we have also generated a fusion protein between RT and the C-terminal part ODC. In present study we eval- uated the immunogenicity of DNA plasmids encoding for HIV-1 RT or RT fused to ODC or ODC degradation signal. 2. Materials and methods 2.1. Proteins and peptides Reverse transcriptase (RT) of HIV-1 HXB-2 (Gen Bank accession number AAB50259) was expressed in Escherichia coli and purified by affinity chromatography as described previously [12]. Peptides 0264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2008.03.070