The fidelity of the product was confirmed by mass spectrometric analysis of tryptic fragments, by the Medical Biomics Centre at SGUL. Fourteen UK captive-bred female cynomolgus macaques (Macaca fascicularis), aged between 4 and 5 years at the beginning of the experiment, were housed in accordance with the Home Office (UK) Code of Practice (1989). Animals were sedated with ketamine hydrochloride prior to procedures. Menstrual cycles were determined by the onset of bleeding. Animals were assigned to experimental groups (Table 1). Immunisation timings varied dependent upon individual menstrual cycles. Gp140
was formulated at 100 μg ml−1 in Carbopol gel as described previously . 1 ml of the mixture was administered via a syringe inserted approximately 2 cm into the selleck kinase inhibitor vagina. For any one cycle of intravaginal inoculation, animals received formulated product on 9 occasions at 2–3 daily intervals during the inter-menses phase of the menstrual selleck chemicals cycle. For systemic immunisation, 100 μg gp140 was mixed with an equal volume of AS01 adjuvant and 0.2 ml injected into each deltoid
muscle. Secretions were sampled using pre-weighed Weck-cel surgical spears (Medtronic Ophthalmics, Jacksonville, FL) placed either on the cervical os or the vaginal wall for 1 min. After reweighing, secretions were eluted from the sponges as described previously . 8 μl of heat inactivated foetal calf serum was added to pooled extracts before freezing aliquots at −80 °C. Total immunoglobulin concentrations from in mucosal eluates were measured by sandwich ELISA. 96-well plates (medium binding, Greiner Bio-One, Stonehouse, UK) were coated with either goat anti-monkey IgG (γ-chain-specific) (KPL, Gaithersburg, USA) or goat anti-monkey IgA (α-chain-specific) (KPL) at 2 μg ml−1. After washing and blocking, as detailed below for antibody ELISA, mucosal eluates were added at dilutions of 1/100
and 1/1000. Bound immunoglobulin was detected by addition of goat anti-monkey IgG (Fc-specific) HRP conjugate (AbD Serotec, Kidlington, UK) or goat anti-monkey IgA (Fc-specific) HRP conjugate. Standard curves were derived using purified rhesus monkey IgG (SouthernBiotech, Birmingham, USA) or purified human IgA (Sigma, UK) and concentrations in mucosal secretions calculated taking into account the dilution factor derived from the weight of sample. Due to the unavailability of purified monkey IgA, results for this isotype were expressed as units (U) ml−1. Anti-gp140 binding antibodies were measured using a standardised ELISA. 96-well plates were coated with 50 μl per well of recombinant CN54gp140 at 5 μg ml−1 in PBS for 1 h at 37 °C. After washing four times in PBS containing 0.05% Tween 20 (PBS-T) reactive sites were blocked by incubation with PBS-T containing 10% foetal calf serum for 1 h at 37 °C. After further washing, serial dilutions of serum or mucosal eluates in PBS-T were added and incubated for 1 h at 37 °C.