1. In vitro–transcribed SFV-LacZ RNA (100 μg) is administered
intramuscularly into BALB/c mice.
2. The immune response is evaluated by monitoring the presence
of IgG antibodies against recombinant β-galactosidase protein
by ELISA 21 days post-injection.
3. Splenocytes are isolated 21 days after immunization and are
restimulated in vitro for 6 days in the presence of Ld-restricted
peptide β-gal 876–884 (1 μg/mL) for monitoring of
β-galactosidase-specific CD8
+ T cell recognition.
Evaluation of tumor protection of mice immunized with in vitro–
transcribed RNA can be performed as follows.
1. Mice are administered intravenously with 5 Â 10
5 CT26.CL25
tumor cells (from mouse colon) and tumor protection is evaluated 21 days postimmunization.
2. The number of pulmonary metastases is counted after 12 days.
3. In case of preestablished tumors, BALB/c mice are injected
intravenously with 1 Â 10
5 CT26.CL25 cells and tumors are
grown for 2 days before immunization with 100 μg
SFV-LacZ RNA.
4. Animal survival is assessed.
3.11.2 Immunization
of Mice with Recombinant
VEE Particles
1. Alphavirus particles (10
6 ) are diluted in PBS and injected subcutaneously into the plantar surface of each footpad of
C57BL/6 mice three times at 2 weeks intervals [37].
2. Vaccinated mice are challenged with 7.5 Â 10
4 B16F10 tumor
cells (from mouse melanoma) intradermally 2 weeks after
immunization for tumor protection evaluation.
3. Therapeutic efficacy is addressed by an initial inoculation of
7.5 Â 10
4 B16F10 tumor cells (either intradermally or intravenously) followed by three weekly vaccinations with alphavirus
particles.
3.11.3 Immunization
of Macaques
with Recombinant VEE
Particles
1. Immunization with 10
10 VEE-EBOV GP focus forming units
(FFUs) is conducted intramuscularly in the quadriceps muscle
of naı ¨ve cynomolgus macaques for vaccine development
against Ebola virus [11].
2. Vaccinated animals are challenged intramuscularly and intranasally with approximately 1000 PFU of Ebola virus and are
monitored closely for at least 28 days.
3.11.4 DNA
Immunization of C57BL/6
Mice with DNA
1. C57BL/6 mice are immunized with 3 μg layered DNA-RNA
plasmid vectors by five weekly intramuscular injections, which
can be enhanced by plasmid-coated gold particles applying
gene gun technology [38].
Alphavirus-Based Antigen Preparation
77
intramuscularly into BALB/c mice.
2. The immune response is evaluated by monitoring the presence
of IgG antibodies against recombinant β-galactosidase protein
by ELISA 21 days post-injection.
3. Splenocytes are isolated 21 days after immunization and are
restimulated in vitro for 6 days in the presence of Ld-restricted
peptide β-gal 876–884 (1 μg/mL) for monitoring of
β-galactosidase-specific CD8
+ T cell recognition.
Evaluation of tumor protection of mice immunized with in vitro–
transcribed RNA can be performed as follows.
1. Mice are administered intravenously with 5 Â 10
5 CT26.CL25
tumor cells (from mouse colon) and tumor protection is evaluated 21 days postimmunization.
2. The number of pulmonary metastases is counted after 12 days.
3. In case of preestablished tumors, BALB/c mice are injected
intravenously with 1 Â 10
5 CT26.CL25 cells and tumors are
grown for 2 days before immunization with 100 μg
SFV-LacZ RNA.
4. Animal survival is assessed.
3.11.2 Immunization
of Mice with Recombinant
VEE Particles
1. Alphavirus particles (10
6 ) are diluted in PBS and injected subcutaneously into the plantar surface of each footpad of
C57BL/6 mice three times at 2 weeks intervals [37].
2. Vaccinated mice are challenged with 7.5 Â 10
4 B16F10 tumor
cells (from mouse melanoma) intradermally 2 weeks after
immunization for tumor protection evaluation.
3. Therapeutic efficacy is addressed by an initial inoculation of
7.5 Â 10
4 B16F10 tumor cells (either intradermally or intravenously) followed by three weekly vaccinations with alphavirus
particles.
3.11.3 Immunization
of Macaques
with Recombinant VEE
Particles
1. Immunization with 10
10 VEE-EBOV GP focus forming units
(FFUs) is conducted intramuscularly in the quadriceps muscle
of naı ¨ve cynomolgus macaques for vaccine development
against Ebola virus [11].
2. Vaccinated animals are challenged intramuscularly and intranasally with approximately 1000 PFU of Ebola virus and are
monitored closely for at least 28 days.
3.11.4 DNA
Immunization of C57BL/6
Mice with DNA
1. C57BL/6 mice are immunized with 3 μg layered DNA-RNA
plasmid vectors by five weekly intramuscular injections, which
can be enhanced by plasmid-coated gold particles applying
gene gun technology [38].
Alphavirus-Based Antigen Preparation
77