Jerome P. Vanderberg

Synchronization of Plasmodium falciparum erythrocytic stages in culture.

Synchronous development of the erythrocytic stages of a human malaria parasite, Plasmodium falciparum, in culture was accomplished by suspending cultured parasites in 5% D-sorbitol and subsequent reintroduction into culture. Immediately after sorbitol treatment, cultures consisted mainly of single and multiple ring-form infections. At the same time, varying degrees of lysis of erythrocytes infected with the more mature stages of the parasite was evident. Approximately 95% of the parasites were in the ring stage of development at 48 and 96 hr after sorbitol treatment-likewise, a high percentage of trophozoite and schizont stages was observed at 24, 72, and 120 hr. D-Mannitol produced similar, selective, lytic effects.

Immunization of man against sporozite-induced falciparum malaria

Based on successful work with nonhuman malarias, an attempt was made to immunize man against mosquito-borne stages of Plasmodium falciparum. Strict ethical guidelines were followed. Mosquitoes carrying sporozoites of P. falciparum were X-irradiated and then fed on volunteers who had not previously been exposed to malaria. The sporozoites were inactivated at a minimum dosage of 15,000 rads, and did not produce adverse reactions in the volunteers. Three volunteers were each exposed during 84 days to 379 infected irradiated mosquitoes, and on day 98 were fed on by nonirradiated mosquitoes heavily infected with homologous strain P. falciparum. One of these men did not develop malaria, and continued to be immunized during the ensuing 217 days with 819 infected mosquitoes. On day 327, when antisporozoite antibody was first demonstrated in his serum by the circumsporozoite precipitation test, he was fed on by nonirradiated mosquitoes carrying homologous strain P. falciparum, and did not develop malaria. Possible causes of his failure to become infected were investigated, and it was concluded that he had become immunized to falciparum sporozoites of that strain. His continued susceptibility to strain-specific falciparum malaria induced by direct blood transfer was demonstrated.

Development of infectivity by the Plasmodium berghei sporozoite.

Studies were done on the development of infectivity during ontogeny of the sporozoite of the rodent malaria parasite, Plasmodium berghei. Populations of sporozoites were separated from the oocysts, the hemocoel, and the salivary glands, with special precautions being taken to avoid cross-contamination between the different populations. The results indicated that populations of salivary gland sporozoites were more than 10,000 times as infective as populations of oocyst sporozoites from the same mosquitoes. The development of this infectivity appears to be asynchronous, in some cases taking place in the hemocoel, while in other cases not occurring until after the sporozoites have invaded the salivary glands. Thus, the development of infectivity seems to be time-dependent rather than site-dependent. There is also a continued increase in sporozoite infectivity during their residence in the salivary gland. The development of infectivity may be associated with other aspects of sporozoite maturation, including changes in their antigenicity and motility.

Anopheles stephensi salivary glands bear receptors for region I of the circumsporozoite protein of Plasmodium falciparum

In the mosquito, Plasmodium sporozoites rupture from oocysts found on the midgut wall, circulate in the hemolymph and invade salivary glands where they wait to be injected into a vertebrate host during a bloodmeal. The mechanisms by which sporozoites specifically attach to and invade salivary glands are not known but evidence suggests that it is a receptor-mediated process. Here we show that the major surface protein of sporozoites, the circumsporozoite protein (CS), binds preferentially to salivary glands when compared to other organs exposed to the circulating hemolymph. In addition, we show that a peptide encompassing region I, a highly conserved sequence found in all rodent and primate Plasmodium CS proteins, inhibits binding of CS to mosquito salivary glands.

Interferon Inducers Protect Mice against Plasmodium berghei Malaria

Injection of mice with two interferon inducers, Newcastle disease virus or statolon, 20 hours after inoculation with Plasmodium berghei sporozoites, prevented or delayed the development of detectable malarial parasitemia and death.

SYNTHESIS AND TRANSFER OF DNA, RNA, AND PROTEIN DURING VITELLOGENESIS IN RHODNIUS PROLIXUS (HEMIPTERA)

1. Histochemical and autoradiographic techniques were applied to Rhodinus prolixus during vitellogenesis.2. Examination of the ovary, fat body, and midgut demonstrated that DNA was synthesized exclusively in the nuclei of these tissues. There appeared to be a transfer of some of the DNA in a partially depolymerized form from the trophic tissues of the ovary to the growing oocyte.3. RNA was synthesized in the nuclei of the ovary, fat body, and midgut, and subsequently was transferred to the cytoplasm of these tissues. Some of the newly synthesized RNA passed from the trophic tissues of the ovary to the enlarging oocyte.4. Protein was found to be synthesized most actively in the follicular epithelial tissues of the ovary, and in the fat body. Evidence of protein synthesis was also found in the other regions of the ovary, and in the midgut. The passage of newly synthesized protein from the follicular epithelium into the oocyte was noted. Synthesis of yolk protein by the oocyte itself appeared to be negligible.

Kinetics of mosquito-injected Plasmodium sporozoites in mice: fewer sporozoites are injected into sporozoite-immunized mice.

Malaria is initiated when the mosquito introduces sporozoites into the skin of a mammalian host. To successfully continue the infection, sporozoites must invade blood vessels in the dermis and be transported to the liver. A significant number of sporozoites, however, may enter lymphatic vessels in the skin or remain in the skin long after the mosquito bite. We have used fluorescence microscopy of Plasmodium berghei sporozoites expressing a fluorescent protein to evaluate the kinetics of sporozoite disappearance from the skin. Sporozoites injected into immunized mice were rapidly immobilized, did not appear to invade dermal blood vessels and became morphologically degraded within several hours. Strikingly, mosquitoes introduced significantly fewer sporozoites into immunized than into non-immunized mice, presumably by formation of an immune complex between soluble sporozoite antigens in the mosquito saliva and homologous host antibodies at the proboscis tip. These results indicate that protective antibodies directed against sporozoites may function both by reducing the numbers of sporozoites injected into immunized hosts and by inhibiting the movement of injected sporozoites into dermal blood vessels.

Further studies on the Plasmodium berghei-Anopheles stephensi-rodent system of mammalian malaria.

Techniques for laboratory maintenance of the Plasmodium berghei-Anopheles stephensi system for rodent malaria are described. The system permits the regular production of sporozoite-induced blood infections in susceptible rodents, as well as the regular attainment of preerythrocytic schizonts in the livers of young white rats. The most sensitive measures of the infectivity of a pool of sporozoites are the time to patency of infected animals, and the percentage of sporozoites which become preerythrocytic schizonts. Three kinds of variability in this infectivity were noted: 1) a variability between different pools of sporozoites, 2) an interspecific variability with tree rats, young white rats, and mice being susceptible in that order, 3) an intraspecific variability, with some animals of a given species being less susceptible than others. Repetitive sporozoite induction of rodent malaria in the laboratory by means of the Plasmodium berghei-Anopheles stephensi system was reported by us in a previous paper (Nussenzweig et al., 1966). Hamsters, tree rats (Thamnomys surdaster), and young white rats were shown to be suitable laboratory hosts, whereas white mice (CF#1 strain) were found to be less readily infected. This difficulty in infecting mice was partially circumvented with the use of an inbred strain of mouse (A/J from Jackson Memorial Laboratories) that was more susceptible to sporozoite-induced infection (Most et al., 1966). Since then, improvements in our technique have enabled us to produce and harvest a greater number of more infective sporozoites. Our experiences with this new system have demonstrated its value in both basic and applied investigations. The sporozoites have been used in chemotherapeutic studies (Most, Herman, and Schoenfeld, 1967), as well as in studies on specific immunity (Nussenzweig et al., 1967) and nonspecific resistance (Nussenzweig, 1967) to sporozoiteinduced infection. The relative ease with which this model may be set up leads us to report our techniques and the characteristics of the sporozoite-induced infections. This paper forms a background for other studies: modification of sporozoites by X-radiation Received for publication 14 May 1968. * This work, contribution no. 384 from the Army Research Program on Malaria, was sponsored by the Commission on Malaria, Armed Forces Epidemiological Board, and was supported by the United States Army Medical Research and Development Command. (Vanderberg et al., 1968), and studies on the use of these irradiated sporozoites as agents for immunization (Nussenzweig et al., 1967). MATERIALS AND METHODS Rearing of mosquitoes Anopheles stephensi were reared in an insectary maintained at 28 C and about 85% relative humidity. Rearing pans containing third and fourth instar larvae were moved to an insectary maintained at 21 C and about 85% relative humidity. The slowing down of development resulting from this drop in temperature allowed us to harvest pupae every other day, instead of daily. Pupae were harvested by means of the cold water technique of Weathersby (1963). Emerged adults were fed 10% glucose solution prior to their infective feeding on hamsters. All mosquitoes were kept on a daily cycle of 16 hr of light and 8 hr of darkness. Infection of hamsters to be used as sources of gametocytes Hamsters used for mosquito feeding were 3 to 5 weeks old. They were infected with an intraperitoneal inoculation of heart blood from a hamster infected with the NK strain of P. berghei berghei. Blood from a donor animal was found to be most useful when the animal was in the rapidly rising phase of an infection in which 20 to 35% of its erythrocytes were parasitized. To guard against senescence of the P. berghei strain only a few hamster to hamster passages of the parasite were used before interposition of a sexual cycle by mosquito passage. More recently we have been fee ng mosquitoes only on hamsters which had been infected with blood from a sporozoite-induced infection. Infection of mosquitoes P ior to their infective blood meal,. mosquitoes were kept at 21 C. The hamsters were anesthetized with an intraperitoneal injection of thialbarbitone sodium (Kemithal?), and placed on top of a cage containing about 250 mosquitoes in the 21 C insectary. The mosquitoes were allowed to feed through

Direct Microscopic Quantification of Dynamics of Plasmodium berghei Sporozoite Transmission from Mosquitoes to Mice

ABSTRACT The number of malaria sporozoites delivered to a host by mosquitoes is thought to have a significant influence on the subsequent course of the infection in the mammalian host. We did studies with Anopheles stephensi mosquitoes with salivary gland infections of Plasmodium berghei sporozoites expressing a red fluorescent protein. After individual mosquitoes fed on an ear pinna or the ventral abdomen of a mouse, fluorescence microscopy was used to count numbers of sporozoites. Mosquitoes allowed to feed on the ear for periods of 3 versus 15 min deposited means of 281 versus 452 sporozoites, respectively, into the skin; this may have epidemiological implications because mosquitoes can feed for longer periods of time on sleeping hosts. Mosquitoes feeding on the ventral abdomen injected sporozoites not only into the skin but also into the underlying peritoneal musculature. Although mosquitoes injected fewer sporozoites into the abdominal tissues, more of these were reingested into the mosquito midgut, probably a consequence of easier access to blood intake from the abdominal area. The most consistent parameter of sporozoite transmission dynamics under all conditions of mosquito probing and feeding was the relatively slow release rate of sporozoites (∼1 to 2.5 per second) from the mosquito proboscis. The numbers of sporozoites introduced into the host by mosquitoes and the transmission efficiencies of sporozoite delivery are multifactorial phenomena that vary with length of probing time, skin site being fed upon, and numbers of sporozoites within the salivary glands.

Plasmodium berghei: Quantisation of sporozoites injected by mosquitoes feeding on a rodent host

Abstract Attempts were made to determine the numbers of viable Plasmodium berghei sporozoites injected by Anopheles stephensi mosquitoes feeding on rodent hosts. Assessment of the numbers injected was made by counting the numbers of exoerythrocytic forms produced by known numbers of infected mosquitoes. The results showed the numbers of viable sporozoites injected to be quite small, on the order of about 1% of the total number of sporozoites in the salivary glands. Mosquitoes with larger numbers of sporozoites in their salivary glands injected more sporozoites. Thus, heavily infected mosquitoes may be more effective vectors. The relative numbers of sporozoites injected by the mosquitoes varied with the host being fed upon. Because of this, it is speculated that host skin factors may be an important determinant of the number of infective sporozoites able to reach the blood after being deposited in the skin.