Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

The overall goal of this procedure is to describe a system to evaluate the physiological initiation of vaccine specific adaptive immunity. This is accomplished first, using reverse genetics to generate live, attenuated influenza vaccines containing a specific immunogenic peptide, and then using a chick embryo to multiply the virus. Next cells are harvested from the lymph nodes of vaccinated mice and fax analysis is used to track dendritic cell migration.

In the next assay, CD eight positive T cells reactive to the immunogenic peptide are labeled and injected into vaccinated mice, followed by fax analysis to measure the proliferation of those cells to analyze the immune response in more detail. A competitive bone marrow chime mirror is made by infusing diptheria toxin receptor expressing bone marrow cells into irradiated congenic mice, which are then exposed to diptheria toxin. After a successful depletion, the mouse is vaccinated and labeled antibodies and tetramers are used to identify CD eight positive T cells.

Recognizing the specific peptide. This method can help answer key questions in the vaccinology field, such as how does a vaccine works at neurological level, which are the main cellular and molecular targets of the vaccine, and most importantly, how we can improve current prophylactic strategies. Demonstrating the procedure will be Sanmen Medina.

Our lab manager and me For this protocol generate a reassorted cold adapted influenza vaccine using the cold adapted strain. A Ann Arbor six 60. As the background coupled with hemagglutinin and a modified neuraminidase of the A PR 8 34 strain.

A portion of the protein stalk sequence of the neuraminidase gene is replaced with a short CDNA sequence encoding the chicken peptide syn fecal. This peptide is highly immunodominant in the H two BMHC class. One restricted response of C 57 black six mice and functions as a traceable element.

To begin plate 1, 000, 002 93 T cells in each well of a six well plate with DMEM containing 10%fetal bovine serum and 1%pen strep. Next, prepare enough plasmid transfection mixture for 100 microliters per well. Combine a microgram of each plasmid and fill the tube with prewarm opti MEM cell medium to 50 microliters in another tube.

Add twice the volume of lipectomy 2000 corresponding to the total quantity of plasmid to be transfected and top the tube off to 50 microliters with opti MEM before adding the transfection mix to the cells. Let it sit for 30 minutes at room temperature. Then add 100 microliters of the transfection mix to each well and move the plate to an incubator for 16 hours.

The next day, change the medium to DMEM with 0.3%BSA and 1%pen strep. Then move the cells to 33 degrees Celsius with 5%carbon dioxide for five hours. After five hours.

Add an overlay of a million influenza permissive MDCK cells in Tripsin supplemented DMEM. Maintain the co cultures for two to three days under the 33 degree Celsius incubation regime to generate and amplify the virus Later, clear out the debris using a five minute 260 G spin. Then collect the supernatants inoculate nine to 10 day old chicken embryo ated eggs under sterile conditions.

To do so, make a hole on the top of the egg shell and add the virus at a 100 x 10 x or one x dilution. Cover the eggshell with melted wax using a cotton swab and incubate the inoculated chicken eggs for three days At 33 degrees Celsius. Later, harvest the Alan Toic fluid from the infected eggs.

Wash the egg shells with 70%ethanol. Open the egg with a very gentle crack in the upper part, and remove the Alan Toic membrane using sterile forceps. Use a 10 milliliter pipette to collect as much fluid as possible.

Typically six to 10 milliliters. Centrifuge the fluid at 260 gs for 10 minutes at four degrees Celsius and transfer the cleared Alan Toic fluid to new tubes. After anesthetizing a donor mouse and surgically accessing the mediastinum, harvest the lymph nodes using curved forceps.

Transfer the lymph nodes to a six well plate containing a milliliter of DMEM. After any connective or fat tissue surrounding the lymph node is removed, secure the lymph node with one needle and break it open with the other needle. This should result in cells bursting out of the lymph node into the media to collect the cells.

First, pass all tissue material through a 70 micron nylon filter to obtain a suspension of cells ready for centrifugation. Resuspend the cell pellet in two milliliters of RBC Lysing buffer. Allow the lysis reaction to go for three minutes at room temperature and end it with a two milliliter addition of ice cold PBS proceed with staining the cells according to the text protocol.

Being sure to use plate flicking to remove and discard the super named before the antibody incubation. Label donor T cells from commercially available TCR transgenic mice incubate 5 million cells in one milliliter of PBS with five micromolar CFSE. Let the incubation go on for 20 minutes at 37 degrees Celsius in the dark.

Then collect the cells at 2 million per 100 microliters of PBS and infuse recipient congenic mice with a 100 microliter bolus via retroorbital sinus injection. After three to five days, euthanize the mice, harvest the mediastinal lymph nodes and prepare a single cell suspension for flow cytometry. As before.

Then identify donor cells by staining the single cell suspensions in an antibody cocktail. For the analysis. Leave the FL one channel of the flow cytometer open to determine the dilution profile of CFSE.

After removing and sanitizing the tibiae and femur of a euthanized mouse, collect them in a dish of DMEM there. Cut bone ends using sharp scissors and flush the bone marrow into the DMEM. Now using standard methods, irradiate six to eight week old female mice using a caesium 1 37 source irradiator two to four hours after irradiating the mice, anesthetize the mice and transplant them with donor bone marrow cells via a retroorbital sinus injection of 2 million cells in 100 microliters of PBS For the next two weeks, supply the mice with 2.5 to five milligrams of Baal per kilogram in their drinking water.

Starting two days before vaccination, administer 50 nanograms of DT diluted in 50 microliters of PBS drop by drop directly to the nostrils of anesthetized mice. Continue this treatment for three days post vaccination. See the text protocol for details to generate the final mouse model.

Vaccinate the mice with recombinant live attenuated influenza using an intranasal injection under anesthesia. Deliver 1000 PFU of vaccine from a 200 microliter pipette in 50 microliters of PBS generation of recombinant live attenuated influenza vaccines can be achieved by transfection of plasmids encoding the eight segments of influenza virus under the control of bidirectional promoters. A cold adapted influenza vaccine usually contains six segments of a cold adapted strain, as well as the HA and NA of the influenza strain of choice, such as H one N one.

Cold adaptation allows for virus restricted replication at 33 degrees Celsius, the temperature of the upper respiratory tract in mice and humans, but does not allow replication in the lower respiratory tract, so it cannot result in pneumonia fusion. PCR was used to replace a conserved region in the stem of the viral neuraminidase with a traceable OVA derived peptide. The traceable peptide model was inoculated into mice between three and six days post vaccination.

The traceable protein syn fecal was detected in the lung draining lymph nodes. Multiparametric flow cytometry was used to quantify vaccine derived antigen presentation in real time. Likewise, infusion of syn fecal specific T-cells allowed quantification of vaccine specific CD eight T-cell responses to evaluate gene specific functions during vaccination.

A mouse model combining DTR technology and competitive bone marrow kyira was utilized. Thus, loss of gene function was targeted to specific cell compartments over the course of immune responses to vaccination. After watching this video, you'll have a good understanding how to analyze molecular and physiological pathways involve in the modulation of vaccine induced immunity in the host.

In addition, this technique will provide new insight in determination of cell targets of the vaccine, thus furthering the valuation of new vaccine platforms.