A NIMALS , IMMUNISATION AND PROTECTION EXPERIMENTS

Female BALB/c or C57BL/6 mice were purchased from Harlan UK, Charles River UK or bred at the University animal facilities and maintained under specific pathogen-freeconditions in individually ventilated cages. Animals were used at 6–9 weeks of age and were age matched within each experiment. All experiments were approvedby a Project License granted by the Home Office (U.K.) and conductedin accordance with local guidelines.

2.10.2 Preparations of frozen salmonella stocks for immunisation

Two hundred ml of LB medium containing the appropriate antibiotics were inoculated 1:100 with a pre-culture and grown at 37 °C; 150 rpm till an OD600nm of 1.4-1.6 was reached. The cultures were centrifuged at 4 °C, 3500xg for 20min and the supernatant was discarded. The pellets were resuspended in 10 ml of LB medium and more medium was added to a volume of 35 ml. Centrifugation was repeated at 4 °C, 3500 xg for 15 min, the supernatant was removed and the pellet was resuspended in 300 μl LB/ 30 % Glycerol. A small sample was diluted to determine OD600nm, the suspension was adjusted to a density equivalent to OD600nm = 100, which corresponds to approximately 10¹⁰ CFU. Suitable aliquots were frozen away in cryotubes at -80 °C. For exact determination of vaccination doses, an aliquot was thawed on ice, serially diluted and dilutions plated on LB agar plates containing appropriate antibiotics. After growth at 37 °C, colonies were counted and CFU numbers calculated.

2.10.3 Purification of outer membrane vesicles

One litre of LB medium with the appropriate antibiotics was inoculated with 10 ml of a preculture grown from a single colony. When the culture reached an OD600nm

between 0.6-0.8, 1 ml of sample was withdrawn and sodium propionate to 50 mM was added to induce the production of AIDA-fusion proteins on the cell surface.

Samples were regularly removed to monitor recombinant protein expression and release into the supernatant. These samples were centrifuged for 3 min at 20800 xg, 100 µl of supernatant were taken and the rest discarded. The pellet was dissolved in 50 µl of 2x protein sample buffer and boiled for 5 min at 95 °C. Pellets and supernatant were frozen at 20 °C until further analysis. After approximately 10 h of induction 50 µg/ ml gentamycin was added for 30 min in order to increase membrane shedding. The cultures were then harvested at 4500 xg for 30 min, the pellets discarded and the supernatant was passed through a 22 µm pore size filter unit. This was followed by ammonium sulphate precipitation using 390 g/ L ammonium sulphate to obtain 60 % saturation. The solution was left to settle over night at 4 °C.

The next morning the precipitate was harvested by centrifugation for 30 min at 11000 xg at 4 °C. The pellets were dissolved in 20 ml PBS and spun again for 15 min at 16000 xg and 4 °C. Afterwards supernatants were transferred into Beckman ultracentrifugation tubes and membrane vesicles were pelleted for 2 h at 100000 xg and 4 °C. To determine dry weights, pellets were dissolved in endotoxin free water and lyophilised. Vesicles were reconstituted in endotoxin free water (HyCult biotechnology bv) to a working concentration of 1 mg/ ml and stored at 4 °C.

2.10.4 Determination of bacterial fitness by colonisation assay

Three mice per group were immunized orally with 1 x 10¹⁰ CFU of a SL3261 vaccine.

After seven days mice were sacrificed and Peyer’s patches removed and placed in a vial containing 1 ml PBS. Subsequently, Peyer’s patches were homogenized between the rough ends of two glass slides and the resulting suspension was transferred into a 2 ml reaction tube. The glass slides were rinsed with an additional 1 ml of PBS.

200 μl of the suspension, 100 μl of 1 % Triton X-100 and 700 μl of PBS were mixed and 100 μl were plated on a selective LB agar plate in duplicates. Out of this dilution

two further dilutions (1:10 and 1:100 respectively) were prepared and also plated on LB agar plates. All plates were incubated overnight at 37 °C and colony numbers were counted the following day.

2.10.5 Infection of mice with L. major and foot pad measurements

L. major promastigotes were grown in 1 x SDM until stationary phase was reached.

Parasites were counted and adjusted to 1 x 10⁷/ 100 µl in PBS and 20 µl (equal 2 x 10⁶ parasites) were injected into the left hind foot pad. Foot pad swelling was measured using a calliper. To determine the overall swelling, the difference in thickness between left infected foot pad and right reference foot pad was calculated.

2.10.6 Determination of L. major burden in murine organs

Mice were sacrificed by cerebral dislocation; organs (spleen, liver, draining lymph node, foot pad) were removed and placed into tubes containing 1 ml of PBS. For processing, organs were forced through a cell strainer using the sterile top end of a 1 ml syringe. Before this treatment, feet were cut several times with scissors to simplify homogenisation. Cell strainers were flushed with 1 ml of PBS and the single cell/ parasite suspension was pipetted back into the collection tube. After homogenisation volumes in all tubes were adjusted to the same level.

For the serial dilution assay, sterile 96 well plates were labelled and filled with 100 µl 1x SDM (supplemented with 20 µg/ ml hygromycin and 50 µg/ ml kanamycin) per well. Homogenates of highly infected foot pads and lymph nodes were pre-diluted 1:100. For serial dilution, 46 µl of cell suspension were added into the wells of the first column in quadruplicates and 46 l were serially transferred resulting in a 1:√10 dilution steps over all 12 wells of a row. The plates were finally sealed with parafilm and incubated at 27 °C for 14 days. Parasite growth was then scored microscopically and parasite load in the infected organs was calculated using the dilution where at least 2 of 4 wells (~37.5 %) of wells were positive (Taswell et al., 1980). The

reciprocal of this dilution was multiplied by the total volume (in multiples of 0.1 ml) to derive the total number of parasites per organ.

2.10.7 Determination of hepatosplenomegally and L. donovani burden in impression smears

Mice were sacrificed, weighed, liver and spleen removed and the weight of both organs was also determined. The organ-body-mass index (BMI) was calculated as the percentage of weight of the single organ to the total body weight of the mouse. To determine parasitic burden in spleen and liver, impression smears from cut organs were prepared on microscopic glass slides. Slides were fixed in methanol and stained with Giemsa. Afterwards the number of parasites per 1000 host cell nuclei was counted using a bright-field microscope and an immersion oil lens. Leishman-Donovan units (LDU) were calculated by multiplying the number of parasites/ 1000 nuclei by the organ weight.

2.10.8 Giemsa staining

Impression smears were briefly fixed in a small volume of methanol. After pouring off excess methanol slides were air dried. Giemsa reagent was prepared in a 50 ml tube by mixing 3.6 ml Sorenson A (9.5g Na2HPO4/L ddH2O), 1.4 ml Sorenson B (9.07g KH2PO4/L dH2O), 45 ml ddH2O and 5.55 ml of a 1:10 dilution of Giemsa stain. The staining solution was mixed carefully and poured into staining baths. Slides were placed upside down in the staining bath and left for 20 to 30 min at room temperature. The stain was then rinsed off under running tap water and slides were allowed to dry.

2.10.9 Blood collection and serum preparation

Mice were bled by submandibular stab incision in accordance to Home Office guidelines. Blood was collected in a 1.5 ml tube and left to clot at 4 °C over night.

The following day, clot and serum were separated by centrifugation at 20800 xg for 15 min and serum was carefully removed and transferred to a new tube, frozen and stored at -80 °C till further use.

2.11 Immunological methods