4. Wash bacterial vectors once and standardize to 0.5 OD 600nm in
PBS, whereas bacterial strains to be used in hybrid vector
formation should be washed once and standardized to 1.0
OD 600nm in 25 mM NaOAc (pH 5.15) (see Note 8).
5. Desiccate the polymer doses that are dissolved in chloroform
and resuspend in 25 mM NaOAc (pH 5.15) prior to equal
volume addition to 1.0 OD 600nm bacterial strains.
6. Incubate hybrid vectors (final 0.5 OD 600nm ) and bacterial vectors in PBS at 22
C for 15 min (see Note 9).
7. Utilize additional PBS to perform further dilutions of both
bacterial and hybrid vectors as desired.
3.7 Hybrid Vector
Immunization
1. Following the preparation described above, the hybrid vector
can then be prepared for animal immunizations. Normalize
dose levels for the hybrid vector to 10
7 by measuring the
OD 600nm of the solution via spectrophotometer and diluting
with PBS, as needed.
2. Prior to the first immunization, collect serum from the mice via
retro-orbital bleeding followed by centrifugation to establish
baseline antibody and cytokine levels.
3. For animal immunizations, utilize CD-1 mice. Immunize mice
in groups of 6–12 using either subcutaneous (SC), intraperitoneal (IP), or intranasal (IN) administration. Administer the
quantity of hybrid vector desired in 200 μL PBS for SC and
IP injections or a 40 μL aspiration for IN delivery. A sham
vaccination with PBS should be included as a negative control
for each administration route. In addition, hybrid vectors containing the background plasmids (i.e., the parent plasmid backbones for CJ05, CJ06, CJ07, pUAB055, and pLF) should be
tested as controls to ensure resulting immune response is
derived from the encoded antigens.
4. After 14 days, collect serum from the mice as described above
to measure antibody and cytokine values after primary
immunization.
5. After collecting the after primary immunization serum, vaccinate the mice with a secondary immunization using the same
conditions.
6. After another 14 days (28 days total), collect serum from the
mice as described above to measure antibody and cytokine
values post-secondary immunization.
3.8 Immunological
Characterization
1. To characterize antibody titers within serum samples, an
enzyme-linked immunosorbent assay (ELISA) is performed
by coating a 96-well Costar high-binding polystyrene plate
with the pneumococcal antigens GlpO, DexB, StkP, or PspA
(10 μg/mL) in tris-buffered saline (TBS) at 4
C overnight.
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Ruiquan Qi et al.
PBS, whereas bacterial strains to be used in hybrid vector
formation should be washed once and standardized to 1.0
OD 600nm in 25 mM NaOAc (pH 5.15) (see Note 8).
5. Desiccate the polymer doses that are dissolved in chloroform
and resuspend in 25 mM NaOAc (pH 5.15) prior to equal
volume addition to 1.0 OD 600nm bacterial strains.
6. Incubate hybrid vectors (final 0.5 OD 600nm ) and bacterial vectors in PBS at 22
C for 15 min (see Note 9).
7. Utilize additional PBS to perform further dilutions of both
bacterial and hybrid vectors as desired.
3.7 Hybrid Vector
Immunization
1. Following the preparation described above, the hybrid vector
can then be prepared for animal immunizations. Normalize
dose levels for the hybrid vector to 10
7 by measuring the
OD 600nm of the solution via spectrophotometer and diluting
with PBS, as needed.
2. Prior to the first immunization, collect serum from the mice via
retro-orbital bleeding followed by centrifugation to establish
baseline antibody and cytokine levels.
3. For animal immunizations, utilize CD-1 mice. Immunize mice
in groups of 6–12 using either subcutaneous (SC), intraperitoneal (IP), or intranasal (IN) administration. Administer the
quantity of hybrid vector desired in 200 μL PBS for SC and
IP injections or a 40 μL aspiration for IN delivery. A sham
vaccination with PBS should be included as a negative control
for each administration route. In addition, hybrid vectors containing the background plasmids (i.e., the parent plasmid backbones for CJ05, CJ06, CJ07, pUAB055, and pLF) should be
tested as controls to ensure resulting immune response is
derived from the encoded antigens.
4. After 14 days, collect serum from the mice as described above
to measure antibody and cytokine values after primary
immunization.
5. After collecting the after primary immunization serum, vaccinate the mice with a secondary immunization using the same
conditions.
6. After another 14 days (28 days total), collect serum from the
mice as described above to measure antibody and cytokine
values post-secondary immunization.
3.8 Immunological
Characterization
1. To characterize antibody titers within serum samples, an
enzyme-linked immunosorbent assay (ELISA) is performed
by coating a 96-well Costar high-binding polystyrene plate
with the pneumococcal antigens GlpO, DexB, StkP, or PspA
(10 μg/mL) in tris-buffered saline (TBS) at 4
C overnight.
470
Ruiquan Qi et al.