Erythrocytic Malaria Growth or Invasion Inhibition Assays with Emphasis on Suspension Culture GIA
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Erythrocytic cycle malaria parasite growth or invasion inhibition assays (GIA) compare the effects of various test and control substances on malaria parasite growth in erythrocytes or invasion into erythrocytes in vitro. Although inhibitions by antimalarial drugs in vitro correlate well with drug protective levels required in vivo, as yet there are too few data to know how well inhibitions by antibodies in vitro correlate with the types and degrees of immune protection in vivo. Antibody-mediated GIA is frequently complicated by parasite strain-specific inhibitions, as well as nonspecific inhibitory factors generated in sera collected or stored under nonoptimal conditions. In this chapter, we describe methods for collecting and processing sera, for using different strains of parasite, and a simplified method for staining parasite DNA with Hoechst dye 33342 before quantitating parasites using ultraviolet (UV)-excited flow cytometry. We also describe a new type of GIA using suspension cultures in a 48-well plate. Critical to this method is enclosing the plate in a gassed, heat-sealed plastic bag, which, being low mass, can easily be rested at a 13.5� angle on a rotor platform (114 rpm with 1-in. displacement) to produce gentle pulsatile waves of media in each well. The suspension GIA, which, relative to the static GIA, increased inhibition by one antibody and decreased inhibition by another Table 1 ), may better simulate in vivo blood flow and may thus better predict in vivo efficacy.
Table 1 An Example of GIA Data a
|
Inhibition per cycle |
|||||
|---|---|---|---|---|---|
|
Suspension |
Static |
||||
|
Isolate |
Anti-A (%) |
Anti-B (%) |
Anti-A (%) |
Anti-B (%) |
|
|
FVO |
52 a |
-7 |
43 |
32 |
|
|
3D7 |
12 |
3 |
4 |
26 |
a Inhibition per cycle (IPC ) compared in suspension GIA and static GIA for two inhibitory antibodies. One set of GIA data (from four 48-well plates, two-cycle cultures, 6% Hct, 0.5 (anti-A) or 1 (anti-B) mg/mL IgG, 150 mL per well) were analyzed by Eq. 3b , IPC = 100% � [1 –(PTF /PCF )1/2 ], where PTF is the final parasitemia in the presence of test antibody and PCF is the final parasitemia in the presence of control antibody. Parasitemias (infected erythrocytes per 100 erythrocytes) were determined by UV flow cytometry of 40,000 Hoechst dye 33342-stained erythrocytes. Initial parasitemias for FVO and 3D7 were 0.04% and 0.02% schizonts, respectively. In the suspension cultures the final control parasitemias for FVO and 3D7 were about 1.6% and 5.3% ring forms, respectively (some variation depending on the source of the control antibody), and in the static cultures the final control parasitemias were about 0.65% and 0.77%, respectively. Standard errors of the mean for the final parasitemias in triplicate wells averaged 0.03% for all but the static 3D7 GIA, which average 0.08%. An inhibition of 52% per cycle corresponded to an inhibition of 77% for the entire GIA. Inhibitions in bold were considered significant and were associated with a p < 0.05 in a two-tailed Students’ t -test when comparing the final parasitemias in the triplicate wells with the test antibody to those with the control antibody. A negative number indicates stimulation— an increased final parasitemia in test compared with control wells. Interpretation : Apparently anti-A inhibited by a mechanism that was as effective, or more, in suspension cultures as it was in static cultures, but was relatively isolate specific. By contrast, anti-B inhibited both isolates about equally well, but inhibited by a mechanism that was effective in the static cultures only.
