Background:  To investigate the immunogenicity and safety of a modified vaccinia Ankara (MVA) vector expressing the HIV-1-LAI Nef gene (MVA-Nef), we conducted a phase-I trial in healthy volunteers.

Methods:  We immunized 14 HIV^­ volunteers (median age 30 years, range 19 to 59) subcutaneously with MVA-Nef at week 0, 4, and 16. In childhood, 2 subjects had received full smallpox vaccination and 2 had received a single smallpox immunization; the other 10 subjects were vaccinia-naive. Nef-specific T cells were monitored by interferon-g (IFN-γ) ELISpot using a set of overlapping peptides, a set of short peptides corresponding to optimal cytotoxic T lymphocyte (CTL) epitopes, Nef-protein, and MVA. Each peptide was analyzed separately using 10⁵ freshly isolated peripheral blood mononuclear cells (PBMC) in triplicate. A positive ELISpot response was defined as ≥ 50 spot-forming units (SFU)/10⁶ PBMC after subtraction of a negative control. In additional stimulation assays, thawed PBMC samples, frozen at the various time-points of the study, were stimulated by HLA-matched peptide pools and outgrowing cells were analyzed after 1 to 2 weeks for recognition of the corresponding peptides.

Results:  MVA-Nef was well tolerated with injection site reactions in all subjects and mild systemic side effects in 11 of 14 subjects. MVA-specific antibodies were detected at baseline at low titres in 4 of 14 subjects (pre-immune). All subjects developed a strong MVA-specific antibody response after the first vaccination. At screening, MVA-specific T cells could be detected in 2 of the pre-immune subjects. All subjects developed MVA-specific T cells at week 18. Nef-specific T cells were detected in freshly isolated PBMC of 7 of the 14 subjects (Nef-specific CTL in 5 subjects, Nef-specific CD4 cells in 4 subjects). ELISpot analysis of peptide stimulated thawed PBMC detected induction of Nef-specific CTL during the vaccination period in 2 additional subjects. Both HLA B13⁺ subjects developed CTL against the HLA B13-restricted immunodominant Nef epitope RQDILDLWI (RI9). RI9-specific CTL were highly cross-reactive for most published HIV-1 variants. Nef-specific antibodies could be induced in two subjects. Both these subjects had also developed Nef-specific CD4-cells.

Conclusions:  MVA-Nef was safe and immunogenic in uninfected subjects and induced a Nef-specific T-cell response in 9 of 14 (64 %) healthy volunteers, whereas Nef-specific antibodies could be detected in 14%. All subjects developed a strong MVA-specific T-cell and antibody response.