Normal AZD6244 concentration control monkey serum was used as a negative control. Standard curves were derived using serum from a macaque immunised with HIV-1W61D gp120 [28].

Antibody titres and concentrations of immunoglobulin were corrected for dilution factor derived from weight of sample/weight of sample + 600 assuming a density of 1 mg μl−1[19]. Neutralising antibody responses were measured against tier 1 and tier 2 HIV-1 envelope-pseudotyped viruses, prepared by transfection of 293T/17 cells, using a standardised luciferase-based assay in TZM.bl cells [29] and [30]. The 50% inhibitory concentration (IC50) titre was calculated as the dilution of serum that gave a 50% reduction in relative luminescence units (RLU) compared to the virus control wells after subtraction of cell control RLUs. Murine leukaemia virus (MuLV) negative controls were included in all assays. Dissected spleen tissue and lymph nodes or marrow washed from the bone were dissociated in RPMI by sieving through a 100 μm mesh and then centrifuged

at 4 °C for 10 min at 400 × g. Supernatant was removed and the pellet resuspended in residual media and washed once more with 10 ml RPMI. Cells were resuspended in 25 ml RPMI and were then filtered through a 50 μm filcon (BD Biosciences, Oxford, UK) before being layered onto Histopaque-1077 (Sigma, UK) and centrifuged at room temperature for 30 min at 1500 × g. Interface cells were collected and viable mononuclear cells counted. Ex vivo amplified Gefitinib datasheet Histone demethylase ELISpot assays were based on the method described by Bergmeier et al. [31]. PVDF membrane plates (Muliscreen HTSIP, Millipore) were treated with 35% ethanol for 1 min, washed three times with sterile PBS and coated with either recombinant CN54 gp140 or KLH (Calbiochem) at 10 μg ml−1 overnight at 4 °C. Following a further 6 washes with PBS-T, reactive sites were Modulators blocked by incubation with RPMI 1640 medium containing 10% FCS and pen/strep for 1 h at room temperature. Freshly recovered tissue MNCs were added to triplicate wells at 1 × 105

and 5 × 105 cells/well and incubated for 24 h at 37 °C in an atmosphere of 5% CO2. After further washing in PBS-T, bound secreted antibody was detected with either goat anti-monkey IgG-HRP (Serotec) diluted 1/2000 or with goat anti-monkey IgA-biotin (Acris) at 1/1000 followed by avidin–HRP (Sigma) diluted 1/2000. Spots were detected by addition of TMB substrate (Sureblue TMB 1-component peroxidise substrate, KPL) and enumerated with a reader. Total IgG and IgA ASC were assayed by the same method using plates coated with goat anti-monkey IgG (γ-chain-specific) (KPL) or goat anti-monkey IgA (α-chain-specific) (KPL) as capture antibodies. Specified analyses were performed using SigmaPlot version 11 software.