Intranasal Immunization with BCG in a Mouse Model

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Quantified Glycerol Stocks of Bacillus Calmette-Guerin (BCG) Danish and Mycobacterium tuberculosis H37Rv

NOTE: All the protocols described were performed under bio-safety level-3 (BSL3) conditions.

1. Thaw frozen glycerol stocks of BCG Danish or H37Rv strains and inoculate 100 μl in 10 ml of 7H9 medium supplemented with Tween-80 0.05% (v/v) and ADC (Albumin, Dextrose, Catalase) 10% (v/v).
2. Incubate under static conditions for one week at 37 °C until the culture is in log-phase growth.
3. Scale up bacterial culture by transferring the 10-ml culture to 200 ml of fresh medium. Incubate for 20 additional days under static conditions.
4. Transfer to 50-ml centrifuge tubes and centrifuge for 15 min at 2500 x g.
5. Discard supernatants and resuspend the bacterial pellet in the residual volume. Add sterile 3-mm-diameter glass beads to each tube (10 – 15 per tube).
6. Vortex vigorously for 1 min in order to dissociate bacterial clumps. Resuspend each pellet in 10 ml of phosphate-buffered saline (PBS) with Tween-80 0.05% (v/v).
7. Leave tubes for 10 min allowing the greatest bacterial aggregates and glass beads to settle down.
8. Recover 9.5 ml of supernatant from each of the 4 tubes containing small clumps and single bacteria and transfer volumes to a single fresh 50-ml centrifuge tube (final recovered volume 38 ml). Centrifuge for 10 min at 400 x g.
9. Recover 35 ml of supernatant (containing mainly single bacteria) in a single fresh 50-ml centrifuge tube and add 15 ml of PBS with glycerol 50% (v/v), obtaining a final glycerol concentration of 15% (v/v).
10. Mix gently and distribute in 1 ml aliquots in appropriate tubes for freezing. Store at -80 °C (one lot provides approximately 50 aliquots of glycerol stock).
11. To quantify glycerol stocks, thaw glycerol the day after freezing and perform seven ten-fold serial dilutions in separate 1.5-ml tubes containing 900 μl of PBS.
12. Plate 100 μl of each dilution in 7H10 solid-agar medium supplemented with ADC 10% (v/v) prepared in 55-mm diameter Petri dishes.
13. Carefully add 3 mm-diameter glass beads (approx. 10 per plate) and gently shake the plate to equally distribute volume over the agar plate. Discard beads and seal the agar plate with parafilm.
14. Incubate for 21 days at 37 °C and determine bacterial concentration by counting colony-forming units (CFU) in the dilutions where single colonies can be accurately distinguished.

2. Mouse Vaccination

1. Thaw a pre-quantified BCG glycerol and dilute it in PBS to prepare two suspensions. Calculate final volume considering the dose per animal is 100 μl for subcutaneous administration (10⁷ CFU per ml for subcutaneous vaccination) and 40 μl for intranasal administration (2.5 x 10⁷ CFU for intranasal vaccination).
2. Use a specialized anesthesia workstation for rodents to anesthetize mice with a mixture of isoflurane and oxygen. Use isoflurane 5% to induce anesthesia and 2% to maintain the mice in the anesthesia chamber. (Other accepted anesthesia method can be used).
3. For subcutaneous vaccine administration fill a 1-ml syringe with a 26 G needle with 1 ml of bacterial suspension (10⁷ CFU/ml). Remove air bubbles.
4. Place an anesthetized mouse on a flat surface in prone position inside a laminar flow hood and inoculate subcutaneously 100 μl (10⁶ CFU/dose) of vaccine in a flank of the mouse back. Return the mouse to the cage and monitor it to ensure that it properly recovers from anesthesia.
5. Change the syringe needle between each mouse administration. Removed air bubbles as described above.
6. For intranasal administration, place an anesthetized mouse in supine position inside the hood.
7. With a micropipette take 20 μl of the suspension with 2.5 x 10⁷ CFU/ml. Place the inoculum drop-by-drop between the two nostrils until the whole volume has been deposited, leaving time between drops to allow the mouse to breathe the volume in.
   NOTE: mice can suffer from respiratory distress during this procedure so they should be left to assimilate each drop prior to administrate the next one to prevent it as much as possible.
8. Re-fill the micropipette with another 20 μl and repeat the operation. If the mouse starts to wake up from the anesthesia after the first inoculation, place it in the anesthesia chamber prior to the second one. Return the mouse to the cage and ensure that it properly recovers from anesthesia.

3. Mouse Intranasal Challenge with H37Rv Strain

1. Thaw a pre-quantified H37Rv glycerol and dilute it in PBS to prepare a bacterial suspension of 2,500 CFU per ml. Calculate final volume considering the dose per animal is 40 μl.
2. H37Rv is inoculated intranasally as described in 2.5 – 2.7. Final challenge dose per mouse is 10² CFU/40 µl.