2. Analysis of the p36p
À Parasite Phenotype During Blood Stage
and Mosquito Stage Development: The analysis of blood stage
development phenotype is undertaken in “asynchronous infections” in Swiss mice and during “standardized synchronized
development in vivo and in vitro” as described [47]. In vitro
studies pertaining to gamete generation, ookinete formation
and fertilization is performed as described [48]. Formation of
oocyst and development of sporozoites are investigated using
Anopheles stephensi. The salivary glands of ten infected mosquitoes are mixed in 300 μl of PBS and the number of sporozoites
per salivary gland is determined after counting the numbers of
sporozoites in duplicate in a cell counter.
3. Analysis of Characteristics of the Infectivity of p36p
À Sporozoites: Female BALB/c and C57BL6 mice of weight 15–20 g
are infected through infected mosquito bites or through intravenous injection of 5 Â 10
4 purified sporozoites. Sporozoites
are collected after dissection of infected mosquito salivary
glands [49]. Each mouse is allowed to be fed by around
20–40 infected mosquitoes for 20 days. Monitoring of the
Blood stage infections is done in Giemsa-stained blood smears
or by analyzing tail blood infected with p36p
À :gfp parasites
through FACS [18] 4–14 day postinfection. Count of the
average circular movement performed by a sporozoite helped
in the estimation of the gliding motility [50]. A total of 4 Â 10
4
sporozoites are centrifuged at 1800 Â g for 10 min onto glass
coverslips coated with 0.02% gelatin in water. Following 2 h
incubation at 37
C staining was performed with anti-CS 3D11
antibody (Ab) for sporozoite and trail visualization. Quantification is done taking into account three independent coverslips
by counting the average number of circles covered by 100 sporozoites. Studies of hepatocyte invasion and traversal are performed in vitro by addition of purified sporozoites to HepG2
cells grown in MEM medium as described [22]. By using a cellimpermeant fluorescent tracer, rhodamine-dextran (1 mg/ml)
[23] parasite-wounded hepatocytes are counted for quantification of cell traversal. The percentage of sporozoites inside
dextran-negative cells is used to determine hepatocyte invasion
as described [51]. Determination of sporozoite development
within HepG2 cells is performed in vitro by staining cells with
different antibodies: anti-PbEXP-1 (detection of a
PVM-resident protein) and anti-HSP90 or anti-HSP70 [52]
(detection of the parasite cytoplasmic heat shock protein 90 or
70). DAPI staining is done to visualize the nuclei. Quantification of trophozoite development is done by counting the
number of trophozoites present in a whole coverslip 24 h
postinvasion.
Live Vaccine Attenuation
351
À Parasite Phenotype During Blood Stage
and Mosquito Stage Development: The analysis of blood stage
development phenotype is undertaken in “asynchronous infections” in Swiss mice and during “standardized synchronized
development in vivo and in vitro” as described [47]. In vitro
studies pertaining to gamete generation, ookinete formation
and fertilization is performed as described [48]. Formation of
oocyst and development of sporozoites are investigated using
Anopheles stephensi. The salivary glands of ten infected mosquitoes are mixed in 300 μl of PBS and the number of sporozoites
per salivary gland is determined after counting the numbers of
sporozoites in duplicate in a cell counter.
3. Analysis of Characteristics of the Infectivity of p36p
À Sporozoites: Female BALB/c and C57BL6 mice of weight 15–20 g
are infected through infected mosquito bites or through intravenous injection of 5 Â 10
4 purified sporozoites. Sporozoites
are collected after dissection of infected mosquito salivary
glands [49]. Each mouse is allowed to be fed by around
20–40 infected mosquitoes for 20 days. Monitoring of the
Blood stage infections is done in Giemsa-stained blood smears
or by analyzing tail blood infected with p36p
À :gfp parasites
through FACS [18] 4–14 day postinfection. Count of the
average circular movement performed by a sporozoite helped
in the estimation of the gliding motility [50]. A total of 4 Â 10
4
sporozoites are centrifuged at 1800 Â g for 10 min onto glass
coverslips coated with 0.02% gelatin in water. Following 2 h
incubation at 37
C staining was performed with anti-CS 3D11
antibody (Ab) for sporozoite and trail visualization. Quantification is done taking into account three independent coverslips
by counting the average number of circles covered by 100 sporozoites. Studies of hepatocyte invasion and traversal are performed in vitro by addition of purified sporozoites to HepG2
cells grown in MEM medium as described [22]. By using a cellimpermeant fluorescent tracer, rhodamine-dextran (1 mg/ml)
[23] parasite-wounded hepatocytes are counted for quantification of cell traversal. The percentage of sporozoites inside
dextran-negative cells is used to determine hepatocyte invasion
as described [51]. Determination of sporozoite development
within HepG2 cells is performed in vitro by staining cells with
different antibodies: anti-PbEXP-1 (detection of a
PVM-resident protein) and anti-HSP90 or anti-HSP70 [52]
(detection of the parasite cytoplasmic heat shock protein 90 or
70). DAPI staining is done to visualize the nuclei. Quantification of trophozoite development is done by counting the
number of trophozoites present in a whole coverslip 24 h
postinvasion.
Live Vaccine Attenuation
351