production, and purification of a recombinant chimeric antigen
containing the detoxified EFn (AA, 1–254), which binds to PA
and allows the entrance of chimeric conserved M2e and HA2
amino acids from influenza. In this step it is necessary to use
bioinformatics tools to find the regions or amino acids conserved
between strains that are exposed to the immune system. The construct contains EFn (1–254)-linker (AAA) and the 3ÂM2e-linker
(GSGGS)-HA2. The chimeric antigen is ligated to an expression
vector and transfected into E. coli. The protein is purified using
denaturing conditions and dialysis to remove contaminants [4, 15].
The second method described here measures vaccinedependent T cell activation and antibody production and their
correlation with protection. The chimeric vaccine without adjuvant
allowed its own translocation into antigen-presenting cells to activate immune responses and induce dual protections against
B. anthracis, Sterne spores, and influenza. Immune cells from
vaccinated mice will be tested against the chimeric construct and
negative controls. Antibodies are used to detect the different conserved antigens and their capacity to neutralize the entrance of
influenza virus into cells by microneutralization assay. Finally, the
protection of mice immunized with the chimeric vaccine and challenge with the three different strains of influenza and B. anthracis
Sterne spores are described.
2 Materials
2.1 Production
and Purification
of the Recombinant
Chimeric Antigen
EFn-3ÂM2e-HA2
1. Synthetic EFn-3ÂM2e-HA2 gene: the anthrax EFn gene
sequence (1–254 aa) was added upstream of three tandem
M2e sequences from A/California/04/2009 (H1N1),
A/Hong Kong/1/1968 (H3N2), and A/Vietnam/1204/
2004 (H5N1) influenza viruses, and the conserved amino
acids in each position were used to create an artificial, centralized, HA2 after alignment of H1N1, H3N2, H5N1, H7N1,
H7N3, and H9N2. Ala-Ala-Ala and Gly-Ser linkers were
designed for insertion between individual gene segments.
2. pET200/D-TOPO (Invitrogen, cat. #K10001).
3. BL21 StarTM (DE3) competent cells (Life Technologies).
4. Kanamycin.
5. IPTG.
6. D(+)-Glucose monohydrate (Fluka Sigma-Aldrich, St. Louis,
MO, USA).
7. Cell lysis buffer (0.5 M NaCl, 40 mM imidazole, and
8 M urea).
8. Elution buffer (20 mM sodium phosphate, 0.5 M NaCl,
500 mM imidazole, pH 7.4).
Vaccine Delivery with a Detoxified Bacterial Toxin
425
containing the detoxified EFn (AA, 1–254), which binds to PA
and allows the entrance of chimeric conserved M2e and HA2
amino acids from influenza. In this step it is necessary to use
bioinformatics tools to find the regions or amino acids conserved
between strains that are exposed to the immune system. The construct contains EFn (1–254)-linker (AAA) and the 3ÂM2e-linker
(GSGGS)-HA2. The chimeric antigen is ligated to an expression
vector and transfected into E. coli. The protein is purified using
denaturing conditions and dialysis to remove contaminants [4, 15].
The second method described here measures vaccinedependent T cell activation and antibody production and their
correlation with protection. The chimeric vaccine without adjuvant
allowed its own translocation into antigen-presenting cells to activate immune responses and induce dual protections against
B. anthracis, Sterne spores, and influenza. Immune cells from
vaccinated mice will be tested against the chimeric construct and
negative controls. Antibodies are used to detect the different conserved antigens and their capacity to neutralize the entrance of
influenza virus into cells by microneutralization assay. Finally, the
protection of mice immunized with the chimeric vaccine and challenge with the three different strains of influenza and B. anthracis
Sterne spores are described.
2 Materials
2.1 Production
and Purification
of the Recombinant
Chimeric Antigen
EFn-3ÂM2e-HA2
1. Synthetic EFn-3ÂM2e-HA2 gene: the anthrax EFn gene
sequence (1–254 aa) was added upstream of three tandem
M2e sequences from A/California/04/2009 (H1N1),
A/Hong Kong/1/1968 (H3N2), and A/Vietnam/1204/
2004 (H5N1) influenza viruses, and the conserved amino
acids in each position were used to create an artificial, centralized, HA2 after alignment of H1N1, H3N2, H5N1, H7N1,
H7N3, and H9N2. Ala-Ala-Ala and Gly-Ser linkers were
designed for insertion between individual gene segments.
2. pET200/D-TOPO (Invitrogen, cat. #K10001).
3. BL21 StarTM (DE3) competent cells (Life Technologies).
4. Kanamycin.
5. IPTG.
6. D(+)-Glucose monohydrate (Fluka Sigma-Aldrich, St. Louis,
MO, USA).
7. Cell lysis buffer (0.5 M NaCl, 40 mM imidazole, and
8 M urea).
8. Elution buffer (20 mM sodium phosphate, 0.5 M NaCl,
500 mM imidazole, pH 7.4).
Vaccine Delivery with a Detoxified Bacterial Toxin
425