Table 5.
Effect of cryopreservation on sporozoite membrane integrity and infectivity in mice inoculated intravenously with the same lot of P. yoelii sporozoites (PySPZ). Infectivity was the number of PySPZ required to infect 50% of BALB/c mice*
| Date | Status of PySPZ | Viability (SMIA) | Number of PySPZ inoculated (IV) | ID50 (number of PySPZ) |
|---|---|---|---|---|
| Oct 2009 | fresh | 96.3% | 24-12-6-3 | 8.9 |
| Dec 2009 | cryopreserved | 72.7% | 200-100-50-25 | 33.1 |
| Dec 2009 | cryopreserved | 68.3% | 200-100-50-25 | 62.1 |
| Jan 2010 | cryopreserved | 67.7% | 400-200-100-50-25 | 103.8 |
| Feb 2010 | cryopreserved | 67.1% | 400-200-100-50-25 | 55.2 |
| Feb 2010 | cryopreserved | 71.6% | 400-200-100-50-25 | 107 |
| Feb 2010 | cryopreserved | 73.9% | 400-200-100-50-25 | 34.5 |
| Mean | cryopreserved | 70.2% | 66.0 | |
| Difference between fresh and cryopreserved PySPZ | 26.1% | 7.4-fold | ||
Freshly dissected, purified P. yoelii sporozoites (PySPZ) were assessed by the sporozoite membrane integrity assay (SMIA) as a measure of viability, and administered to BALB/c mice by intravenous (IV) injection. The remaining PySPZ from the same lot were cryopreserved, thawed at six different time points, assessed for viability by SMIA, and administered IV to mice. To provide data for calculation of the number of PySPZ that infected 50% of mice (ID50 calculated using an exponential association model y = a(1-e−bx)) (CurveExpert version 1.4) with fresh and cryopreserved PySPZ, groups of five mice each received PySPZ in de-escalating doses as indicated, and their infection status was determined by assessing Giemsa-stained blood smears 7–14 days after inoculation. The viability by SMIA of purified, cryopreserved PySPZ was reduced 26.1% as compared with fresh, purified PySPZ. The cryopreserved PySPZ were 7.4-fold less infective than fresh PySPZ as it took 7.4 times more cryopreserved PySPZ to achieve 50% infection of mice.