South Africa showed good tolerance, but only modest immune
responses [24]. In another clinical trial, VEE particles expressing
the carcinoembryonic antigen (CEA) were administered at intramuscular doses of 4 Â 10
7 IU to 4 Â 10
8 IU in four cycles every
third week [25]. Repeated administration induced clinically relevant CEA-specific T cell and antibody responses and longer survival
was seen in patients with CEA-specific T cell responses. Another
phase I trial was conducted with VEE particles expressing the
prostate-specific membrane antigen (PSMA) in patients with castration resistant metastatic prostate cancer (CRPC) [26]. Administration of five doses of 0.9 Â 10
7 IU or 3.6 Â 10
7 IU of
VEE-PSMA was well tolerated although only weak PSMA-specific
responses were detected, most likely due to suboptimal dosing.
The use of alphavirus vectors describing the steps from plasmid
DNA preparation to virus particle production and immunization
studies including cell culture techniques, virus purification and
concentration and titer determination methods is presented
below. All methods describe here are for SFV, although they are,
in most cases, similarly applicable for SIN and VEE.
2 Materials
2.1 Reagents
and Equipment
1. Restriction endonucleases NruI, SpeI.
2. 0.8% agarose gel.
3. Gel electrophoresis apparatus.
4. Phenol–chloroform–isoamyl alcohol 25:24:1 (v/v/v).
5. 3 M Sodium acetate, pH 4.8.
6. 95% and 70% (v/v) ethanol.
7. 10Â SP6 Buffer: 400 mM HEPES, pH 7.4, 60 mM magnesium acetate, 20 mM spermidine.
8. 10 mM m
7
G(5
0 ) ppp (5
0 ) G sodium salt (Roche Molecular
Biochemicals).
9. 50 mM Dithiothreitol (DTT).
10. rNTP Mix: 10 mM rATP, 10 mM rCTP, 10 mM rUTP,
5 mM rGTP.
11. 10–50 U/μL RNase inhibitor.
12. 10–20 U/μL SP6 RNA polymerase (Amersham Pharmacia
Biotech).
13. RNase-free water (DEPC treated).
14. Phosphate buffered saline (PBS).
15. Trypsin–ethylenediamine tetraacetic acid (EDTA): 0.25% trypsin, 1 mM EDTA Â 4 Na.
66
Kenneth Lundstrom
responses [24]. In another clinical trial, VEE particles expressing
the carcinoembryonic antigen (CEA) were administered at intramuscular doses of 4 Â 10
7 IU to 4 Â 10
8 IU in four cycles every
third week [25]. Repeated administration induced clinically relevant CEA-specific T cell and antibody responses and longer survival
was seen in patients with CEA-specific T cell responses. Another
phase I trial was conducted with VEE particles expressing the
prostate-specific membrane antigen (PSMA) in patients with castration resistant metastatic prostate cancer (CRPC) [26]. Administration of five doses of 0.9 Â 10
7 IU or 3.6 Â 10
7 IU of
VEE-PSMA was well tolerated although only weak PSMA-specific
responses were detected, most likely due to suboptimal dosing.
The use of alphavirus vectors describing the steps from plasmid
DNA preparation to virus particle production and immunization
studies including cell culture techniques, virus purification and
concentration and titer determination methods is presented
below. All methods describe here are for SFV, although they are,
in most cases, similarly applicable for SIN and VEE.
2 Materials
2.1 Reagents
and Equipment
1. Restriction endonucleases NruI, SpeI.
2. 0.8% agarose gel.
3. Gel electrophoresis apparatus.
4. Phenol–chloroform–isoamyl alcohol 25:24:1 (v/v/v).
5. 3 M Sodium acetate, pH 4.8.
6. 95% and 70% (v/v) ethanol.
7. 10Â SP6 Buffer: 400 mM HEPES, pH 7.4, 60 mM magnesium acetate, 20 mM spermidine.
8. 10 mM m
7
G(5
0 ) ppp (5
0 ) G sodium salt (Roche Molecular
Biochemicals).
9. 50 mM Dithiothreitol (DTT).
10. rNTP Mix: 10 mM rATP, 10 mM rCTP, 10 mM rUTP,
5 mM rGTP.
11. 10–50 U/μL RNase inhibitor.
12. 10–20 U/μL SP6 RNA polymerase (Amersham Pharmacia
Biotech).
13. RNase-free water (DEPC treated).
14. Phosphate buffered saline (PBS).
15. Trypsin–ethylenediamine tetraacetic acid (EDTA): 0.25% trypsin, 1 mM EDTA Â 4 Na.
66
Kenneth Lundstrom