Sporozoite-induced Infections Research Articles

Observations on the Biological Nature of Plasmodium vivax Sporozoites

The relapsing malaria parasites are characterized by the production of sporozoites with varying potential for exoerythrocytic development. Some sporozoites develop soon after introduction to produce mature schizonts and merozoites that initiate the erythrocytic stage infection. Relapsing hypnozoite forms are characteristic of some strains of Plasmodium vivax and are more apt to develop late than early with many time intervals in between. Studies in Saimiri monkeys suggest another type of sporozoite-induced infection. With the Salvador I strain of P. vivax, early developing exoerythrocytic schizonts apparently release parasites with different levels of virulence for these monkeys, ranging from those producing high-level parasitemia to a more abundant avirulent form. The induction of low-density avirulent infections requires the development of more sensitive detection methods for the evaluation of sporozoite vaccines.

Studies of a Chloroquine-Resistant Strain of Plasmodium vivax from Papua New Guinea in Aotus and Anopheles farauti s.l.

AMRU-1, a chloroquine-resistant strain of Plasmodium vivax from Papua New Guinea, was established in Aotus monkeys as a potential model for chemotherapeutic studies. The course of infection in 16 untreated, splenectomized, Colombian, and Panamanian animals was reasonably reproducible with parasitemias being maintained above 1,000/mm3 for an average of 13.7 days. Moderate to high parasitemias (1,200-68,800/mm3) were recorded in these monkeys, with a mean maximum parasitemia of 20,587.5/mm3. The sibling species Anopheles farauti 1, A. farauti 2, and A. farauti 3 were infected with gametocytes of this strain; these gametocytes completed the sporogonic phase, producing sporozoites that were infective to Aotus monkeys. Maximum infections in mosquitoes occurred 3-4 days prior to the peak parasitemia in the monkeys. Using these 3 species of Anopheles, sporozoite-induced infections were obtained in monkeys in 9 of 15 attempts with an average prepatent period of 32 days. The findings show that a suitable model system could be developed to assess the activity of antimalarial drugs against the blood stages and possibly the liver stages of the AMRU-1 strain.

Evaluation of WR250417 (A Proguanil Analog) for Causal Prophylactic Activity in the Plasmodium cynomolgi-Macaca mulatta Model

The Plasmodium cynomolgi-Macaca mulatta model has been used to test the antimalarial activity of new drugs for both radical cure and casual prophylaxis. The proguanil analog WR250417 (also known as PS-15) was evaluated for causal prophylactic activity in rhesus monkeys infected with P. cynomolgi bastianelli. Four monkeys were orally dosed with 40 mg/kg/day of WR250417 over three days (-1, 0, and +1). Sporozoite-induced infection of P. cynomolgi was initiated on day 0 with 1 x 10(6) sporozoites to each monkey. Compound WR250417 extended the prepatent period from an average of 8.5 days for controls (n = 2) to a mean of 18.3 days (range 18-19 days, n = 4) for drug-treated monkeys. Analysis of plasma drug concentrations by high-performance liquid chromatography showed that the monkeys converted the WR250417 to its putative principal active metabolite WR99210 (a dihydrotriazine). These findings demonstrate that WR250417 and its principal metabolite do not prevent primary infection by P. cynomolgi.

Human interferon-gamma protects rhesus monkeys against sporozoite-induced Plasmodium cynomolgi malaria infection.

Earlier we reported that prophylactic treatment with recombinant human interferon gamma (rHuIFN-gamma) for a prolonged duration completely suppressed experimental infection with Plasmodium cynomolgi B sporozoites in rhesus monkeys. We now show that reducing the rHuIFN-gamma treatment to three administrations given close to the time (on days -1, 0, and +1) of infection was sufficient for complete protection. Animals initially protected by rHuIFN-gamma treatment are susceptible to reinfection. Studies are in progress to understand the precise mechanism of protection afforded by HuIFN-gamma against sporozoite-induced malaria infection.

Pathogenicity of Haemoproteus meleagridis (Haemosporina: Haemoproteidae) in Experimentally Infected Domestic Turkeys

Sporozoite-induced experimental infections of Haemoproteus meleagridis produced a moderate to severe myositis and significant effects on weight gain and growth in domestic turkey poults. Pathological effects occurred in both low- and high-dose infections (4,400 and 57,500 sporozoites, respectively). Low-dose birds weighed significantly less than controls at 3 wk postinfection (PI) when peripheral parasitemia reached a peak and had significantly shorter tarsometatarsal lengths at both 1 and 3 wk PI. High-dose birds were significantly lighter and smaller than control and low-dose birds throughout the course of the 8-wk study. Infected birds were not anemic in spite of high parasitemias that often exceeded 50% of circulating erythrocytes. The most serious pathological effects occurred prior to patency and were associated with development of megaloschizonts in skeletal muscle. Microscopic lesions in 4 high-dose birds that died between 19 and 22 days PI were characteristic of a severe, acute hemorrhagic myositis. Megaloschizonts were surrounded by a hemorrhagic inflammatory infiltrate composed of macrophages, heterophils, giant cells, and red blood cells. Muscle fibers adjacent to megaloschizonts were swollen, hyaline, and contained prominent calcium deposits. Other observations included enlargement of the spleen, deposition of pigment in macrophages of the lung and spleen, and secondary bacterial and fungal infections in the intestine and lungs. Necrotic and calcified muscle fibers and degenerating megaloschizonts were still present at 8 wk PI when the experiment ended. Our results demonstrated significant pathological changes in H. meleagridis-infected domestic turkeys that were associated primarily with preerythrocytic stages of development.

The real difficulties for malaria sporozoite vaccine development: nonresponsiveness and antigenic variation

Antibody against the circumsporozoite coat (CS) protein and cytolytic T lymphocytes (CTL), probably also specific for the CS protein, can block sporozoite-induced malaria parasite infection. These mechanisms of immunity define current strategies for sporozoite vaccine development. In this article, Michael Good and colleagues emphasize that these approaches demand that the CS protein is immunogenic in all members of the population. Unfortunately, the CS protein is not immunogenic for T cells in up to 40% of adults in a region of Africa where malaria is endemic, and studies in mice suggest that immunogenicity is under the control of the immune response (lr) genes. Furthermore, the regions of the CS protein that are immunogenic for T cells are variant. Both the variation and the poor immunogenicity may be the result of selection pressures acting against hepatocytes bearing immunogenic parasites, which will be recognized and killed by CTL.

Attenuation of the Virulence of the M Strain of Plasmodium cynomolgi during Prolonged Multiplication in Splenectomized Rhesus Monkeys

The primary data in this report showed that prolonged multiplication of the M strain of Plasmodium cynomolgi in splenectomized rhesus monkeys led frequently to emergence of parasites whose virulence for monkeys with intact spleens was markedly attenuated while that for splenectomized monkeys was unimpaired. Expressions of attenuation regularly included reductions in the height of the peak parasitemia and in some instances failure to induce infection with inocula 1,000-fold those infective for splenectomized monkeys. Attenuation of virulence, once established, was maintained through as many as 17 serial passages in intact monkeys and more than 100 such transfers in splenectomized monkeys. Not only asexual blood stages, but also sexual stages (gametocytes) carried the attenuated characteristic, as indicated by its ready passage through mosquitoes. Studies in monkeys with sporozoite-induced infections showed that the persisting exoerythrocytic stages did not participate in the attenuation phenomenon; for when erythrocytic parasites of reduced virulence were cleared from the blood by either immune processes or chemotherapy, they were replaced upon relapse with parasites of unimpaired virulence. A major effort to determine whether splenectomized monkeys carried plasma or erythrocyte factors responsible for displays of attenuated virulence was nonproductive. An intact spleen was the essential element in these displays, for they disappeared immediately upon removal of this organ. The attenuation phenomenon probably reflects selection of spontaneously occurring mutants with limited capacity to escape normal splenic clearance mechanisms and unimpaired or even enhanced capacity to multiply in splenectomized monkeys.

Inhibition of development of exoerythrocytic forms of malaria parasites by gamma-interferon.

A specific DNA probe was used to study the effect of recombinant rat, mouse, and human gamma-interferon (gamma-IFN) on the course of sporozoite-induced malaria infections. In mice and rats infected with sporozoites of Plasmodium berghei, mouse and rat gamma-IFN's strongly inhibited the development of the exoerythrocytic forms in the liver liver cells of the hosts, but not the development of the erythrocytic stages. The degree of inhibition of the exoerythrocytic forms was proportional to the dose of gamma-IFN administered, but was independent of the number of sporozoites used for challenge. A 30 percent reduction in the development of exoerythrocytic forms in rat liver was achieved when 150 units (about 15 nanograms of protein) of rat gamma-IFN were injected a few hours before sporozoite challenge; the reduction was 90 percent or more with higher doses of gamma-IFN. The effect was less pronounced if the gamma-IFN was administered 18 hours before or a few hours after challenge. Human gamma-IFN also diminished the parasitemia in chimpanzees infected with sporozoites of the human malaria parasite Plasmodium vivax. The target of gamma-IFN activity may be the infected hepatocytes themselves, as shown by in vitro experiments in which small doses of the human lymphokine inhibited the development of exoerythrocytic forms of Plasmodium berghei in a human hepatoma cell line. These results suggest that immunologically induced interferon may be involved in controlling malaria infection under natural conditions.

Inhibition of lymphocyte invasion by sporozoites and the transformation of trophozoite infected lymphocytes in vitro by serum from Theileria annulata immune cattle.

Serum from calves immune to Theileria annulata inhibited sporozoite-induced infection and transformation of peripheral blood leucocytes (PBL) into macroschizont infected lymphoblastoid cells in vitro. Invasion by sporozoites was suppressed by some sera, especially that obtained after challenge, but in many instances suppression of infection could be attributed only to retardation of development of PBL infected with intracellular trophozoites into proliferating macroschizont infected cells. Suppression occurred both in cultures of PBL from susceptible calves and in cultures of PBL from T. annulata immune calves. More uninfected than infected blast cells were found both in cultures of PBL from susceptible calves and in cultures of PBL from immune animals. Results suggested that proliferation of uninfected PBL from susceptible calves was initiated by the transformation of sporozoite infected cells, whilst proliferation of uninfected PBL from immune calves also incorporated a component which was a mitotic response of sensitised lymphocytes to sporozoites (i.e. parasite antigen). Thymidine incorporation by cultures was a measure of proliferation of both uninfected and infected blast cells. It could be used, therefore, to monitor parasite development only where proliferation of total blast cells was directly related to proliferation of infected blasts, as in cultures of PBL from susceptible calves. This study suggests that resistance of cattle to tick derived challenge with sporozoites is mediated initially by serum factors acting both on sporozoites and on the development of intracellular trophozoites to schizonts; that the proliferation of uninfected lymphocytes in naive cattle infected with tropical theileriosis is a direct response to the proliferation of Theileria infected cells and that the proliferation of uninfected lymphocytes in Theileria immune cattle is a combined response to the proliferation of Theileria infected cells and parasite antigen.

Antimalarial activity of floxacrine (HOE 991) II: Studies on causal prophylactic and blood schizontocidal action of floxacrine and related dihydroacridinediones against Plasmodium yoelii and P. berghei.

The causal prophylactic and blood schizontocidal action of six dihydroacridinediones were assessed in mice using the Plasmodium yoelii 17X strain in sporozoite-induced infections and the P. berghei K 173 strain in blood-induced infections respectively. A remarkable relationship was shown between the action on pre-erythrocytic forms and on blood forms of malaria parasites. A trifluoromethyl group in the para (4—) position of the 3-phenyl ring of the 7-chloro-10-hydroxy-3,4-dihydroacridine-1,9-(2H,10H)-dione (compound Hoe 991: floxacrine) proved the most favourable substituent of a group of electronegative substituents concerning antimalarial activity. The CPD50/CD50 of Hoe 991 against pre-erythrocytic/blood forms of P.yoelii/P. berghei was 0·37 mg kg−1 (X 1)/1·7 mg kg−1 (X5) after the intraperitoneal (ip)/subcutaneous (sc) route. As demonstrated by compound Hoe 990 double substitution in the 3-phenyl ring with 2-chloro-, 4-trifluoromethyl- caused a marked residual action. The causal prophylactic effect (CP...

I. The Characteristics of Untreated Sporozoite-Induced and Trophozoite-Induced Infections

Abstract This report summarizes the results of a longitudinal study of the characteristics of untreated infections with trophozoites and sporozoites of the M, B, and Ro strains of P. cynomolgi. The pre-initial-peak phases of infections with trophozoites of these strains were remarkably similar. These phases of sporozoite-induced infections were also similar except that infections with the B and Ro strains had a shorter incubation period and reached the initial peak more promptly than infections with the M strain. The post-initial-peak phase of the primary attack in infections with the M strain was significantly shorter than that phase of infections with either the B or Ro strain and was characterized by parasitemias of lesser intensity and fewer secondary peaks. The total duration of infections with trophozoites of either the M or B strain from onset of patency to self-cure was markedly shorter than the duration of infections with sporozoites of these strains. The duration of infections with sporozoites of the B strain was significantly longer than the duration of infections with the M strain. Monkey-to-monkey variations in the features of the post-initial-peak phase of infections, including the frequency of secondary attacks and the total duration of disease, were substantial. Although malaria morbidity was a common feature of primary attacks, the incidence of malaria deaths was low in infections with all three strains.

Plasmodium cynomolgi infections in the rhesus monkey.

The studies on infections with Plasmodium cynomolgi recorded in this Supplement had their origins in the World War II search for drugs that at well-tolerated doses would effect radical cure of naturally acquired infections with Plasmodium vivax. At its onset, this search rested almost completely upon screening of new agents for activities against trophozoite-induced and sporozoite-induced infections with P. gallinaceum in chickens or P. cathemerium in canaries and trophozoite-induced infections with P. lophurae in ducks. Compounds that exhibited promising activities in these systems, and appropriate levels of tolerability in rats, dogs, and rhesus monkeys, were evaluated in human volunteers for capacities to prevent or cure infections induced with sporozoites of P. vivax. None of the agents selected on the basis of activities against the avian malarias exhibited such capabilities.

II. Responses of Sporozoite-Induced and Trophozoite-Induced Infections to Standard Antimalarial Drugs

Abstract Quinine, quinacrine, chloroquine, amodiaquine, chlorguanide, and pyrimethamine, administered at fractions of their maximum tolerated doses, regularly cured established infections with trophozoites of the M strain of Plasmodium cynomolgi. Chloroquine and pyrimethamine were similarly effective against such infections with the B and Ro strains. On the other hand, none of these agents, administered at maximum tolerated doses, effected cure of established infections with sporozoites of the M strain or prevented infections when delivered from time of sporozoite challenge to the end of the incubation period. Chloroquine and pyrimethamine were equally ineffective against established infections or challenges with sporozoites of the B and Ro strains. In contrast, pamaquine, pentaquine, isopentaquine, and primaquine, administered in well tolerated doses, either alone or in combination with quinine, effected cure of established infections with sporozoites of the M strain and, delivered alone, provided full protection against infections with sporozoites. Primaquine, administered in combination with chloroquine, was similarly curative of established infections with sporozoites of the M, B, and Ro strains. Administered alone, it was fully protective against infections with the B and Ro strains. These responses to standard drugs, replicas of those obtained in infections with the New Guinea Chesson strain of Plasmodium vivax in human volunteers, provided the underpinning for use of infections with various strains of P. cynomolgi in the rhesus monkey in the search for more effective prophylactic, radical curative, and suppressive antimalarial drugs.

Suppressive and Causal Prophylactic Activity of Floxacrine in Various Avian Malaria Models

Floxacrine, a 7-chloro-10-hydroxy-3(4-trifluoromethylphenyl)-3, 4-dihydroacridine-1,9-(2H, 10H)-dione and various standard malarials were found to be distinctly less active against avian malaria parasites than against rodent or nonhuman primate malaria parasites. Especially the suppressive action against asexual stages of P. gallinaceum, P. praecox and P. cathemerium was markedly reduced in comparison with that ascertained in mice infected with P. berghei. Similar results were observed in sporozoite-induced infections of P. gallinaceum and P. cathemerium respectively. Expressed as quotient of calculated causal prophylactic activity (CPD50) floxacrine was 42 and pyrimethamine 54 times more active against P. berghei yoelii than P. gallinaceum. The blood schizontocidal effect of floxacrine proved superior to that of mefloquine, chloroquine and mepacrine, whereas the causal prophylactic activity was slightly inferior to that of pyrimethamine but superior to that of primaquine. In vitro floxacrine revealed no effect against sporozoites of P. gallinaceum even at concentrations of 100 mcg/ml.

Primaquine liposomes in the chemotherapy of experimental murine malaria.

Due to a significant reduction of toxicity, 100% curative (causal prophylactic) doses of primaquine diphosphate entrapped in liposomes (60 and 70 mg/kg body weight) can be administered in a single intravenous injection to treat sporozoite-induced Plasmodium berghei infections of mice.Although drug activity is not enhanced as compared to free drug, a simplified experimental therapy scheme could be developed, which may facilitate comparative pharmacokinetic studies of various drug delivery systems.

The dual role of macrophages in the sporozoite-induced malaria infection. A hypothesis

Macrophages were thought to act as a barrier to sporozoite entry in liver cells. Evidence is presented here for a possible dual role of macrophages under different sets of conditions. Before activation macrophages might act as transporting agents of sporozoites to the parenchymal cells of the liver. Activated macrophages however might take the opposite role of preventing parasite entry. Various possible mechanisms involved are reviewed.

The Characteristics of Plasmodium cynomolgi Infections in Various Old World Primates *

Abstract This report summarizes the results of a comparative study of the major features of untreated infections with the B strain of Plasmodium cynomolgi in Macaca mulatta, M. irus (fascicularis), M. nemestrina, M. speciosa (arctoides), and Cercopithecus aethiops. The investigation included: delineation of the courses of trophozoite-induced and sporozoite-induced infections in these Old World primates; evaluation of their efficiencies in transmitting the above plasmodium through Anopheles freeborni; and assessment of host reactions to infection, with particular attention to impacts on erythroid elements of peripheral blood. In all important respects, the parasitic courses of infections in M. mulatta and C. aethiops (Kenyan origin) were identical, as were the reactions of these hosts to disease processes. M. mulatta was slightly more efficient than C. aethiops in transmitting P. cynomolgi through A. freeborni; however, this favored position may have been purchased by experimental design. The features of infections in M. irus, M. nemestrina, and M. speciosa differed significantly from those in M. mulatta. M. irus responded to challenge with trophozoites or sporozoites in a highly reproducible manner, but exhibited peak parasitemias and a long series of parasitic waves (recrudescences and/or relapses) of much lower intensities than were encountered in M. mulatta. M. nemestrina responded to such challenges in diverse manners. A few subjects exhibited parasitic courses almost identical with those encountered in M. irus. The majority exhibited extremely low level parasitemias of great persistence. These diverse parasitic courses were associated with specific physical features of this monkey. M. speciosa failed to support infections following inoculation with either trophozoites or sporozoites. Sporozoite challenges did lead to transient, extremely low level parasitemias, indicating that tissue stage schizogony had taken place. The unusual features of infections in M. nemestrina and M. speciosa could be of substantial interest to those concerned with factors which determine invasion of erythrocytes by plasmodia. Demonstration that untreated infections in M. mulatta and C. aethiops are essentially identical is of far more immediate importance. If responses of infections in these hosts to standard antimalarial drugs are also identical, the way is open to substituting C. aethiops for M. mulatta in studies on the biology and therapy of infections with P. cynomolgi, particularly in the search for radical curative drugs. Such substitution would meet a critical need at a time when access to feral M. mulatta is restricted.

Immunity to Sporozoite-Induced Malaria Infection in Mice

Abstract The cellular basis of immunity to sporozoites was investigated by examining the effect of immunization of T and B cell-deficient C57BL/6N × BALB/c AnN F1 (BLCF1) mice compared to immunocompetent controls. Immunization of T cell-deficient (ATX-BM-ATS) BLCF1 mice with x-irradiated sporozoites did not result in the generation of protective immunity. The same immunization protocols protected all immunocompetent controls. In contrast, B cell-deficient (μ-suppressed) BLCF1 mice were protected by immunization in the majority of cases. The absence of detectable serum circumsporozoite precipitins or sporozoite neutralizing activity in the μ-suppressed mice that resisted a sporozoite challenge suggests a minor role for these humoral factors in protection. These data demonstrate a preeminent role for T cells in the induction of protective immunity in BLCF1 mice against a P. berghei sporozoite infection.

Sporozoite Transmission of Falciparum Malaria (Burma-Thau. Strain) from Man to Aotus Monkey *

A sporozoite-induced infection of Plasmodium falciparum (Burma-Thau. strain) was established in splenectomized Aotus trivirgatus which received prior treatment with dlmethionine as a dietary supplement. This is the first recorded instance of infection of an Aotus monkey with falciparum sporozoites of human origin.

Antimalarial quinones for prophylaxis based on a rationale of inhibition of electron transfer in Plasmodium.

Knowledge of the biochemistry of Plasmodium is emerging as a new field. Previous studies showed that the parasite apparently requires electron transfer for energy, and techniques to study such energy mechanisms are available. The discovery of the existence of coenzyme Q(8) in Plasmodium implies an indispensable functionality for this redox entity in the electron transfer of the parasite, as coenzyme Q(n) similarily functions in other forms of life. Effective antimalarial activity in prophylaxis has been demonstrated in sporozoite-induced infections by Plasmodium gallinaceum in chicks by several representatives of 7-alkylmercapto-6-hydroxy-5,8-quinolinequinones. The absence of toxicity in this assay even at greatly elevated dosage underscores the achievement of selectivity and safety to the host for the potential utilization of antimetabolites of coenzyme Q(n) as medicinals. Seven new 7-alkylmercapto-6-hydroxy-5,8-quinoline-quinones were synthesized. The structural variations of the 7-alkylmercapto group in relationship to the antimalarial activities reveal substantial differences in biological activities, which can reflect molecular specificities of enzyme sites and which are not evident from the deceptively minor structural differences in the alkylmercapto groups. These analogs of coenzyme Q(8) having effective antimalarial activity are known to inhibit mammalian coenzyme Q(n) enzymes, and could be useful in elucidation of the basic electron transfer mechanisms of Plasmodium.