2. Mice are inoculated with 1 Â 10
5 B16F10 tumor cells 1 week
after the last immunization and monitored for tumor growth
for at least 3 weeks.
3.11.5 DNA
Immunization of BALB/c
Mice with Recombinant
VEE Particles
1. SFV plasmid DNA vectors expressing membrane proteins PrM
and E of Murray Valley encephalitis virus (MVE) are diluted in
saline to a concentration of 1 mg/mL and doses of
100–125 mg DNA and are injected intramuscularly into
BALB/c mice [39].
2. Immunized mice are challenged intraperitoneally with
1.3 Â 10
8 PFU of MVE and signs of encephalitis are observed
for 21 days. Alternatively, SPF mice are immunized intramuscularly with 100 mg DNA into multiple sites in the hind leg
muscles and are boosted after 21 days.
3. Mice are intracranially challenged with 1000 TCID 50 of MVE
2 weeks after the final immunization and are monitored for
signs of encephalitis for 21 days.
3.12 Future
Directions
and Potential for Other
Applications
Antigen production applying alphaviruses has been straight forward and successful. The clear advantages of alphavirus-based
approaches relate to easy and rapid vector preparation, the selfamplifying RNA replication leading to enhanced transgene expression, and for example in the case of DNA-based delivery, the
requirement of 100- to 1000-fold lower dose concentrations for
immunization [13]. Likewise, it has been demonstrated that
64 times less self-amplifying RNA (1.25 μg) compared to synthetic
mRNA (80 μg) was required to provide protection in immunized
mice [12]. In addition to alphavirus vector development including
less cytotoxic mutants [40, 41] (see Note 6) and translation
enhancement signals [42], delivery issues have been addressed
particularly for RNA by lipid nanoparticle encapsulation [43]. In
this context, self-amplifying VEE RNA encapsulated in lipid nanoparticles enhanced immunogenicity compared to delivery of unformulated RNA providing a novel approach for improved vaccine
development.
4 Notes
1. To ensure the production of both good quality and high quantity of in vitro–transcribed RNA, it is recommended to use
plasmid DNA preparations of high purity.
2. Optimization of in vitro transcription yields can be achieved by
titration of the CAP analogue m
7 G(5
0 )ppp(5
0 )G concentration
and the use of an appropriate transcription buffer. Although
commercially available buffers might generate high RNA
78
Kenneth Lundstrom
5 B16F10 tumor cells 1 week
after the last immunization and monitored for tumor growth
for at least 3 weeks.
3.11.5 DNA
Immunization of BALB/c
Mice with Recombinant
VEE Particles
1. SFV plasmid DNA vectors expressing membrane proteins PrM
and E of Murray Valley encephalitis virus (MVE) are diluted in
saline to a concentration of 1 mg/mL and doses of
100–125 mg DNA and are injected intramuscularly into
BALB/c mice [39].
2. Immunized mice are challenged intraperitoneally with
1.3 Â 10
8 PFU of MVE and signs of encephalitis are observed
for 21 days. Alternatively, SPF mice are immunized intramuscularly with 100 mg DNA into multiple sites in the hind leg
muscles and are boosted after 21 days.
3. Mice are intracranially challenged with 1000 TCID 50 of MVE
2 weeks after the final immunization and are monitored for
signs of encephalitis for 21 days.
3.12 Future
Directions
and Potential for Other
Applications
Antigen production applying alphaviruses has been straight forward and successful. The clear advantages of alphavirus-based
approaches relate to easy and rapid vector preparation, the selfamplifying RNA replication leading to enhanced transgene expression, and for example in the case of DNA-based delivery, the
requirement of 100- to 1000-fold lower dose concentrations for
immunization [13]. Likewise, it has been demonstrated that
64 times less self-amplifying RNA (1.25 μg) compared to synthetic
mRNA (80 μg) was required to provide protection in immunized
mice [12]. In addition to alphavirus vector development including
less cytotoxic mutants [40, 41] (see Note 6) and translation
enhancement signals [42], delivery issues have been addressed
particularly for RNA by lipid nanoparticle encapsulation [43]. In
this context, self-amplifying VEE RNA encapsulated in lipid nanoparticles enhanced immunogenicity compared to delivery of unformulated RNA providing a novel approach for improved vaccine
development.
4 Notes
1. To ensure the production of both good quality and high quantity of in vitro–transcribed RNA, it is recommended to use
plasmid DNA preparations of high purity.
2. Optimization of in vitro transcription yields can be achieved by
titration of the CAP analogue m
7 G(5
0 )ppp(5
0 )G concentration
and the use of an appropriate transcription buffer. Although
commercially available buffers might generate high RNA
78
Kenneth Lundstrom