5. Coomassie blue.
2.3.3 T Cell Immune
Responses by Detection
of the Stimulated T Cell
Population
1. Mouse immunized spleens.
2. Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml
penicillin, and 100 μg/ml streptomycin (DMEM-10).
3. 100-μm nylon mesh cell strainers
4. ACK lysis buffer.
5. 10 ng of PMA/100 ng ionomycin.
6. M2, 3ÂM2e-HA2, PA.
7. M2e-region peptides from A/New York/348/03 (H1N1)
virus.
8. HA2 region peptides from A/California/04/2009 (H1N1)
virus.
9. Antibodies: anti-CD3–FITC, CD4–PerCP-Cy5.5, CD8–PE,
CD8–V500, and FoxP3–PE (Table 1).
10. BD FACSCanto II flow cytometer (BD Biosciences).
11. FLOWJO v10 software (FlowJo, Ashland, OR).
2.3.4 Tetramers
and Staining
1. I-A(d)/SLLTEVETPIRNEWGS–phycoerythrin (MHC Class
II) and H-2K(d)/VETPIRNEW–allophycocyanin (MHC
Class I), obtained through the NIH Tetramer Core Facility at
Emory University (Atlanta, GA).
2. DMEM-10.
3. Spleen cells.
4. 100-μm nylon mesh cell strainers
5. Anti-CD3–Alexa Fluor 700, CD4–APC, CD8–phycoerythrin,
and CD3–FITC (BD Biosciences, Table 1).
2.3.5 Lethal Challenge
with Influenza Viruses or
B. anthracis Sterne Spores
1. EFn-3ÂM2e-HA2-vaccinated mice.
2. 2 Â 10
5 PFU of live A/PR/8/34 virus and 1 Â 10
5 PFU of live
A/CA/4/09 virus (ATCC, Manassas, VA).
3. B. anthracis Sterne spores.
4. Weight scale and thermometer.
3 Methods
3.1 Production
and Purification
of the Recombinant
Chimeric Antigen
EFn-3ÂM2e-HA2
A synthetic chimeric gene was generated by combining the EFn
gene (1–254 residues); three tandem M2e sequences from influenza subtypes A/California/04/2009 (H1N1), A/Hong Kong/
1/1968 (H3N2), and A/Vietnam/1204/2004 (H5N1, H3N2,
and H5N1) (Fig. 1a) to increase the density and variation of M2e
epitopes; and a highly centralized HA2 gene sequence.
Vaccine Delivery with a Detoxified Bacterial Toxin
427
2.3.3 T Cell Immune
Responses by Detection
of the Stimulated T Cell
Population
1. Mouse immunized spleens.
2. Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml
penicillin, and 100 μg/ml streptomycin (DMEM-10).
3. 100-μm nylon mesh cell strainers
4. ACK lysis buffer.
5. 10 ng of PMA/100 ng ionomycin.
6. M2, 3ÂM2e-HA2, PA.
7. M2e-region peptides from A/New York/348/03 (H1N1)
virus.
8. HA2 region peptides from A/California/04/2009 (H1N1)
virus.
9. Antibodies: anti-CD3–FITC, CD4–PerCP-Cy5.5, CD8–PE,
CD8–V500, and FoxP3–PE (Table 1).
10. BD FACSCanto II flow cytometer (BD Biosciences).
11. FLOWJO v10 software (FlowJo, Ashland, OR).
2.3.4 Tetramers
and Staining
1. I-A(d)/SLLTEVETPIRNEWGS–phycoerythrin (MHC Class
II) and H-2K(d)/VETPIRNEW–allophycocyanin (MHC
Class I), obtained through the NIH Tetramer Core Facility at
Emory University (Atlanta, GA).
2. DMEM-10.
3. Spleen cells.
4. 100-μm nylon mesh cell strainers
5. Anti-CD3–Alexa Fluor 700, CD4–APC, CD8–phycoerythrin,
and CD3–FITC (BD Biosciences, Table 1).
2.3.5 Lethal Challenge
with Influenza Viruses or
B. anthracis Sterne Spores
1. EFn-3ÂM2e-HA2-vaccinated mice.
2. 2 Â 10
5 PFU of live A/PR/8/34 virus and 1 Â 10
5 PFU of live
A/CA/4/09 virus (ATCC, Manassas, VA).
3. B. anthracis Sterne spores.
4. Weight scale and thermometer.
3 Methods
3.1 Production
and Purification
of the Recombinant
Chimeric Antigen
EFn-3ÂM2e-HA2
A synthetic chimeric gene was generated by combining the EFn
gene (1–254 residues); three tandem M2e sequences from influenza subtypes A/California/04/2009 (H1N1), A/Hong Kong/
1/1968 (H3N2), and A/Vietnam/1204/2004 (H5N1, H3N2,
and H5N1) (Fig. 1a) to increase the density and variation of M2e
epitopes; and a highly centralized HA2 gene sequence.
Vaccine Delivery with a Detoxified Bacterial Toxin
427