P22. Construct, design and immunogenicity of a minicircle DNA vector encoding a short HIV peptide

Sofia Stenler1, Lena Hansen2, Jorma Hinkula3, Stefan Petkov2, Karl Ljungberg2, Karin E Lundin4, Pontus Blomberg1, Britta Wahren2
Affiliates: 1Vecura, Karolinska University Hospital, SE‐14186 Huddinge, Sweden 2Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-17177 Solna, Sweden 3Department of Clinical and Experimental Medicine, Linköping University, SE-581 83 Linköping, Sweden 4Department of Laboratory Medicine, Karolinska Institutet, SE‐14186 Huddinge, Sweden

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Introduction
We have constructed a novel minicircle vector (MC) encoding a short immunogenic HIV-1 peptide but devoid of bacterial sequences, and compared its immunogenicity to a conventional plasmid vector. The MC is known for its prolonged expression and increased robustness compared to conventional plasmids.
The peptide short peptide is T20, Fuzeon Enfuvirtide;S a well-established antiviral drug against HIV. As T20 blocks virus entry into the CD4+ cell by mimicking part of the viral transmembrane protein, it could also serve as an antigen against HIV. The T20 aminoacid sequence contains the epitope for a broadly neutralizing antibody, 2F5.

Methods
A novel MC construct was cloned, encoding the 108 base pairs (bp) long sequence of T20 flanked by the CMV promoter and other regulatory sequences. The entire MC encompasses 1 134 bp. MC were produced in E.Coli, by recombination of the parental plasmid into one MC containing only the expression cassette and one rest plasmid containing Ori and antibiotics resistance, see Fig 1. The MC was then purified and tested for expression in vitro. Mice were immunized intradermally by electroporation with the MC encoding T20 alone, or with either MC or plasmid encoding T20 in combination with a HIV-DNA plasmid mix encoding Env subtyps A, B, C. Sera were tested in anti-T20 and anti-gp140 ELISA. Spleens were collected and used in IFN-γ ELISpot assays.

Results
Expression of peptides as short as 36 aminoacids is not trivial, in nature they are often produced as multimers which are then cleaved. Thus we first tested expression in vitro, and through western blotting we could confirm peptide production from the MC.
In mice, the MC induces production of antibodies to the T20 peptide which hat harbors the epitope 2F5. MC given together with the HIV-DNA plasmid mix strongly enhanced end point titers against both gp140 C and T20 (Fig 2 A,B). Sera from mice immunized with HIV-DNA-Env plasmids and the T20 plasmid resulted in increased antibody titers also against other sites of the gp160 HIV protein. The majority of these latter antibodies were not T20-specific.
Spleenocyte analysis showed T20-specific cellular responses for the mice receiving the MC or the plasmid as adjuvants, see Fig 2 C. Neither the peptide nor the full length HIV-DNA-Env plasmids alone elicited any response.

Conclusion
We demonstrate that giving a small plasmid based construct encoding a short immunogenic peptide results in a clear-cut induction of antibodies to the T20 epitope that competes with binding of the T20-specific 2F5 monoclonal. Using the MC-T20 as adjuvant to the HIV-DNA plasmid mix strongly enhanced end point titers against both gp140 C and T20 compared to HIV-DNA plasmid mix alone. Together with HIV-DNA plasmid mix, pT20 and MC-T20 but not T20 peptide elicited T20-specific IFN-γ responses.

P22-sofia_stenler