The overall goal of this procedure is to study immune responses in the lungs following tuberculosis immunization, including the protective efficacy of the vaccine against an intranasal challenge and the local vaccine-induced lung immune response. This methodology can help answer our key questions in the tuberculosis vaccine felt, including identification of the defensive lung immuno responses tuckered by pulmonary vaccines for optimal lung immune protection. One benefit of this methodology is that the lung immune response from its animal is individually assessed in parallel with protective efficacy.
allowing the identification of the specific immune parameters that correlate with protection. For subcutaneous administration of the BCG vaccine, first, fill a one milliliter syringe equipped with a 26 gauge needle with one milliliter of the BCG vaccine suspension, and remove the air bubbles. Then place an anesthetized mouse in the prone position inside a Laminar flow hood, and subcutaneously inoculate the animal with 100 microliters of bacteria in a rear flank.
For intranasal administration, load a micro-pipette with 20 microliters of the BCG suspension, and place the anesthetized mouse in the supine position inside the hood. Administer the inoculum dropwise between the two nostrils until the entire 20 microliter volume has been deposited, leaving time between the drops to allow the mouse to inhale the suspension. Then, reload the micro-pipette with another 20 microliters of bacteria and repeat the administration.
For intranasal challenge with the H37Rv microbacterium tuberculosis strain, at the appropriate experimental time point post-vaccination, inoculate the animals with intranasal administration of 40 microliters of the H37Rv bacterial suspension as just demonstrated. To acquire the bronchoalveolar lavage or ball sample, first use sterile forceps and scissors to expose the trachea. Then make a small incision in the trachea, taking care not to excise the tissue completely, and insert a cannula connected to a sterile 1 mL syringe, containing 800 microliters of ice-cold PBS.
When the cannula is in place, carefully fill the lungs with the PBS, then slowly pull back on the plunger to recover at least 500 to 600 microliters of lavage fluid. Dispense the ball into a 1.5 mL tube on ice, and confirm that the suspension is colorless, indicating a lack of blood contamination. Store samples on ice until ball processing.
To determine the bacterial load and immune response in the lungs, fix the mouse with needles in the supine position on a flat and disinfected surface inside the Laminar flow hood, and make an incision in the upper skin over the chest. Then, peel back the skin to expose the thoracic area, and cut open the lungs. Use sterile scissors and forceps to harvest the lungs and heart, and place the organs on a clean surface.
Then, remove the heart, trachea, and connective tissue. Place each lung in a different 1.5 mL tube with 1 mL of de-ionized water on ice for downstream determination of bacterial load and cellular immune responses. When compared to the subcutaneous route, the intranasal route of vaccination confers a much greater protective efficacy in the lungs.
Analysis of a representative number of colonies from the intranasal BCG vaccine group by PCR specific for the RD9 genome region demonstrates that all of the colonies provide a fragment of 0.4 kilo-base-pairs that corresponded to H37Rv. Intranasal BCG triggers higher IL17 and interfere on gamma production in the lungs, revealing a correlation between the protective efficacy conferred by the intranasal BCG vaccination and the vaccine induced immune response in the lungs prior to challenge with a higher concentration of both the total and the H37Rv purified protein derivate-specific IgA concentration in the lavage samples observed for the intranasal vaccine recipients. Further analysis of cytokine producing cells in the lungs following the tuberculosis challenge revealed that whereas interfere on gamma producing cells were detected in all infected groups, IL17A producing CD4+cells were found only in the intranasal BCG group, demonstrating a significant correlation between the presence of IL17 and lung-bacterial load reduction.
Following this procedure, it is possible to compare the local lung immuno-responses in the use of biodiverse tuberculosis vaccines as well as different routes of administration and time points after challenge. In addition, this methodology can be used to evaluate the immunicity in the used by vaccination after challenge. Which could allowed for the identification of the immuno responses that correlate with protection.
Importantly, working with Mycobacterium Tuberculosis requires BCL-free lab facilities, and the appropriate precautions should always be taken when performing this procedure.
