Mice (n = 4–8 per group) were prime-boost immunised i.n./i.m. with 1 × 107 plaque forming units (PFU) rFPV followed by 1 × 107 PFU rVV expressing HIV-1 antigens and IL-13Rα2 or IL-4C118 antagonist as described in Table 1 under mild methoxyfluorane anaesthesia two weeks apart. Similarly groups of

mice were used as unimmunised controls. Immediately prior to delivery the viruses were diluted in phosphate buffered saline (PBS) and sonicated 20–30 s to obtain an homogeneous viral suspension, intranasal rFPV was given in a final volume of 20–25 μl and i.m. rVV were delivered, 50 μl per quadriceps. To evaluate CD8 T cell mediated protective immunity, 6 weeks post booster vaccination, immunised and unimmunised mice were challenged intranasally with 75–100 PFU of influenza virus PR8 expressing the KdGag197–205 epitope of HIV as described previously [23]. Body weight was monitored BI 6727 datasheet for 9–10 days after challenge. The attenuated recombinant influenza virus PR8-KdGag197–205

incorporates the H2-Kd restricted immuno-dominant epitope AMQMLKETI [32] into the influenza virus neuraminidase stalk, constructed as described by Cukalac et al. [39]. Intra-nasal challenge of naïve BALB/c Torin 1 datasheet (H2-Kd) mice with PR8-KdGag197–205 induces significant weight loss, followed by weight gain as the mice recover from a mild flu, over a 10 day period. The cells infected with PR8-KdGag197–205 present the MHC-I restricted HIV-Gag epitope, and in HIV Gag immunised mice CD8+ CTL specific for HIVGag197–205 will kill the infected cells limiting replication and dissemination of the recombinant influenza virus significantly reducing weight loss. The ability to maintain body weight about specifically at peak infection (4–7 days) is considered a measure of CD8+ T cell mediated protective immunity not antibody immunity. To measure systemic and mucosal T cell responses mice were euthanized at different time intervals (2 and 8 weeks) post-boost immunisation, and 10 days post influenza-KdGag197–205 challenge; spleen, genito-rectal nodes (GN) or iliac lymph nodes and Peyer’s patch (PP) were

removed and cell suspensions prepared in complete (5% FBS) RPMI as described previously [20], [40] and [41]. Allophycocyanin-conjugated KdGag197–205 tetramers were synthesised at the Bio-Molecular Resource Facility at The John Curtin School of Medical Research (BRF/JCSMR), ANU. 2–5 × 106 splenocytes or mucosal lymphocytes were stained with anti-CD8-FITCα antibody (Biolegend, USA) and Allophycocyanin-conjugated KdGag197–205 tetramer at room temperature and analysed as described previously [20], [40] and [42]. All the appropriate controls were performed and the background tetramer counts in naïve mice were found to be between 0.05 and 0.5% in spleen, 0.02–0.05% in mucosal tissue and GN. Also following KdGag197–205 tetramer staining the dissociation assays were performed as described before [21] and [43].