Michael J. Stewart

Antigenic diversity in Thai Plasmodium vivax circumsporozoite proteins

Extensive polymorphism in the central repetitive portion of circumsporozoite (CS) proteins has been found in the simian malarias Plasmodium cynomolgi [1] and Plasmodium knowlesi [2] and in the related human malaria Plasmodium vivax [3]. CS protein tandem repeat sequences are known to vary between Plasmodium species, but the above examples show intra-specific variation and antibodies raised against particular strains may not cross-react with others. This is a potentially serious problem for vaccines based on raising immune responses to a single form of a polymorphic protein. Little quantitative information exists about the distribution of CS protein variants in parasite populations although Rosenberg et al. [3] found that at least two non-crossreacting variants occurred in sporozoites developing in mosquitoes after feeding on vivax malaria patients in Thailand. One isolate had a CS gene encoding a tandemly repeating nonapeptide which differed from the previously reported repeat at 6/9 positions. Since the non-repetitive aminoand carboxyterminal domains are almost completely conserved, it seems likely that all P. vivax CS proteins are derived from a common ancestral gene. However we have very little information about the extent and distribution of allelic variation in

Induction of hepatic inflammatory response by Plasmodium berghei sporozoites protects balb/c mice against challenge with Plasmodium yoelii sporozoites

BALB/c mice are about 2,000 times less susceptible to sporozoites of Plasmodium berghei than to Plasmodium yoelii. Associated with this is the innate cellular response mounted after injection with P. berghei. Host inflammatory cells do not normally attack P. yoelii during their development as exoerythrocytic forms (EEFs) in the liver. We used P. berghei sporozoites to induce host inflammation that might act against developing P. yoelii EEFs. Mice injected with P. berghei sporozoites followed 1 hr later with P. yoelii had a 58% reduction in P. yoelii EEFs. To establish whether this was due to events that occurred before vs. after invasion of hepatocytes by P. yoelii sporozoites, mice received P. yoelii sporozoites that were allowed to invade for 1 hr before subsequent injection with P. berghei; these mice showed minimal reduction in P. yoelii EEFs. Thus, most of the deleterious effects of P. berghei sporozoites appear to have been directed against P. yoelii sporozoites prior to their invasion of hepatocytes. Plasmodium yoelii that had already invaded were relatively unaffected. Further timing experiments showed that this effect was induced only by viable P. berghei sporozoites, which may thus induce rapid changes in sinusoid physiology leading to host resistance against P. yoelii sporozoites.

Antibody-facilitated macrophage killing of Trypanosoma musculi is an extracellular process as studied in several variations of an in vitro analytical system.

Antibody‐facilitated macrophage (MP) destruction of Trypanosoma musculi involves ingestion and intracellular degradation of the parasites. It is likely, however, as we show here, that death of the trypanosomes is extracellular and it is the corpses that are ingested by MPs. We have utilized both peritoneal MPs and a cloned line (WLG 5) of mouse MPs to analyze the killing of T. musculi. Both types of MP were more effective when activated by interferon‐γ (IFN‐γ) rather than lipopolysaccharide (LPS). When activated by both, LPS diminished the killing activity stimulated by IFN‐γ, perhaps by changing the spectrum of lysins/toxins released by the MPs. Nitric oxide (NO) was found to be toxic for T. musculi and to be responsible, in part, for MP killing of the parasites. Although antibody and complement in concert caused lysis of T musculi, complement was not required for MP killing of the parasites. In the course of this investigation, we developed an in vitro system, involving line 5 MPs and plasma from infected mice containing resident parasites, that should prove satisfactory for detailed analyses of the mechanisms of the antibody‐dependent, cell‐mediated cure of T. musculi infection. J. Leukoc. Biol. 56: 636–643; 1994.

In vitro Invasion of Host Cells by Plasmodium berghei Sporozoites in Serum-Free Medium

The development of techniques for the in vitro invasion of Plasmodium berghei sporozoites into continuous cell lines of WI38 human embryonic lung fibroblasts (Hollingdale et al., 1981, Science 213: 1021-1022) and HepG2 human hepatoma cells (Hollingdale et al., 1983, American Journal of Tropical Medicine and Hygiene 32: 685-690) has made it possible to investigate many aspects of sporozoite-host cell interactions during the process of sporozoite invasion. We recently used this in vitro procedure to demonstrate a direct correlation between the motility of sporozoites and their ability to invade both of these cell lines (Stewart et al., 1986, Infection and Immunity 51: 859-864). Agents and procedures that inhibited sporozoite motility invariably prevented sporozoites from invading. We had previously demonstrated that sporozoites suspended in culture medium containing serum or albumin undergo active motility when these sporozoites are pipetted onto a microscope slide and examined by phase-contrast microscopy (Vanderberg, 1974, Journal of Protozoology 21: 527-537); sporozoites thoroughly rinsed and suspended in protein-free medium are immotile. Because active sporozoite motility appears to be necessary for sporozoite invasion into target host cells in vitro (Stewart et al., 1986, loc. cit.), we were interested to see whether the absence of serum in the medium would affect sporozoite invasiveness into HepG2 and WI38 cells. Sporozoites were obtained from the salivary glands of P. berghei-infected mosquitoes that had fed on infective hamster blood 18 days previously (Vanderberg, 1977, Experimental Parasitology 42: 169-181). The salivary glands were pooled in ice-chilled Medium M199 (GIBCO Laboratories, Grand Island, New York) and triturated in a glass homogenizer to release the sporozoites. After a low-speed centrifugation step to pellet the mosquito debris, the sporozoites in the supernatant were thoroughly washed in Medium M199 and kept on ice until used. We found it necessary to wash the sporozoites because the concentrate from triturated mosquitoes appeared to contain an undefined soluble component that induced a degree ofsporozoite motility. For our invasion studies, we inoculated 3,000 sporozoites previously rinsed with medium (with or without 10% fetal bovine serum [FBS]) onto 12-mm-diameter target cell monolayers incubated in media (Eagles Minimal Essential Medium [MEM], GIBCO, Grand Island, New York) supplemented with or without FBS (Stewart et al., 1986, loc. cit.). These sporozoites effectively invaded both HepG2 and WI38 cells when the cultured cells were incubated in medium containing serum (data not shown). However, sporozoites can invade HepG2 cells effectively even in the absence of serum, although the absence of serum significantly inhibits the entry of sporozoites into WI38 cells (Table I). That P. berghei sporozoites invade HepG2 cells more effectively than WI38 cells confirms previous observations (Hollingdale et al., 1983, loc. cit.; Stewart et al., 1986, loc. cit.). These results appeared to be directly contradictory to our hypothesis that sporozoite motility is necessary for invasiveness, because serum albumin is normally required for the induction of sporozoite motility. To determine whether the sporozoites that invaded cultured cells in medium lacking serum are actively motile when in contact with the surface of cultured cells, we inoculated M199-rinsed sporozoites onto con-

Dextran Density Gradient Purification of Plasmodium Sporozoites

Several procedures have been described for isolating relatively pure preparations of Plasmodium sporozoites from homogenates of infected mosquitoes. These have included gradient centrifugation (on continuous or discontinuous diatrizoate gradients), column purification (with ion-exchange or lectin affinity columns), and filtration through pores of defined size (review and comparative assessment of these techniques in Vanderberg, 1980, In The in vitro cultivation of the pathogens of tropical diseases, W.H.O. Tropical Diseases Research Series, 3, Schwabe & Co., Basel, pp. 77-89). We now report a new gradient procedure, which yields better and more consistent results than any previously described. Plasmodium berghei sporozoites were obtained from Anopheles stephensi mosquitoes infected 18-22 days previously (procedures as in Vanderberg, 1977, Experimental Parasitology 42: 169-181). Generally, 3 separate batches of 100200 mosquitoes were gently homogenized for 2 min with 1.5 ml Medium M199 (GIBCO Labs., Grand Island, New York) in an ice-chilled mortar. The pooled homogenates were transferred to a 15-ml centrifuge tube, and additional medium added to make a total of 10 ml. The preparation was centrifuged at 20 g for 6 min, after which the supernatant suspension was transferred to a 12-ml glass Sorvall centrifuge tube. The pellet, containing residual sporozoites, was returned to a mortar and gently ground with 2 ml M199 for 2 min; the resulting homogenate was transferred to a 15-ml centrifuge tube, additional M199 to