The role of Plasmodium knowlesi in the history of malaria research.

In recent years, a malaria infection of humans in South East Asia, originally diagnosed as a known human-infecting species, Plasmodium malariae, has been identified as a simian parasite, Plasmodium knowlesi. This species had been subject to considerable investigation in monkeys since the 1930s. With the development of continuous culture of the erythrocytic stages of the human malarial parasite, Plasmodium falciparum in 1976, the emphasis in research shifted away from knowlesi. However, its importance as a human pathogen has provoked a renewed interest in P. knowlesi, not least because it too can be maintained in continuous culture and thus provides an experimental model. In fact, this parasite species has a long history in malaria research, and the purpose of this chapter is to outline approximately the first 50 years of this history.

Plasmodium knowlesi infections in a small number of non-immune natural hosts (Macaca fascicularis) and in rhesus monkeys (M. mulatta).

The natural host of Plasmodium knowlesi is the kra monkey, Macaca fascicularis, but this parasite, initially mistaken for P. malariae, is now infecting humans in some areas of Southeast Asia. Here we present data from experiments performed in the 1970s in which sera from a few naive M. fascicularis, taken in the course of a first infection, exhibited rapidly rising inhibition of in vitro replication of P. knowlesi. The results were compared with sera from P. knowlesi-infected rhesus monkeys that usually die if left untreated.

Development of malaria blood-stage vaccines: learning from mosquitoes.

If current methods of vaccine development for malaria continue to result in vaccines with only relatively limited degrees of protection, what is the alternative? Here, a totally different approach to blood-stage vaccine research is suggested, focusing on malarial immunity as it develops in macaque monkeys, but using methodology already well established in mosquito research.

T-cell depletion and immunity to malaria in HIV-infections.

Although early reports on HIV and malaria in co-infected subjects indicated little apparent interaction between the two infections, more recent investigations have found evidence for HIV increasing the risk from malaria. Conversely, increased viral load in susceptible cells occurs in malaria-infected people. However, the overall pattern of results is still somewhat confusing and contradictory. While morbidity from malaria may be greater in HIV-positive patients and in several reports the mortality risk is also higher, major increases in blood-stage parasitaemias that one might expect are not generally observed. The results of surveys are summarized and discussed in the context of what is known of malaria and HIV immunology in the light of recent data from humans as well as animal models.

P25 and P28 proteins of the malaria ookinete surface have multiple and partially redundant functions.

The ookinete surface proteins (P25 and P28) are proven antimalarial transmission-blocking vaccine targets, yet their biological functions are unknown. By using single (Sko) and double gene knock-out (Dko) Plasmodium berghei parasites, we show that P25 and P28 share multiple functions during ookinete/oocyst development. In the midgut of mosquitoes, the formation of ookinetes lacking both proteins (Dko parasites) is significantly inhibited due to decreased protection against lethal factors, including protease attack. In addition, Dko ookinetes have a much reduced capacity to traverse the midgut epithelium and to transform into the oocyst stage. P25 and P28 are partially redundant in these functions, since the efficiency of ookinete/oocyst development is only mildly compromised in parasites lacking either P25 or P28 (Sko parasites) compared with that of Dko parasites. The fact that Sko parasites are efficiently transmitted by the mosquito is a compelling reason for including both target antigens in transmission-blocking vaccines.

Inhibition of the mosquito transmission of Plasmodium berghei by Malarone (atovaquone-proguanil).

Sera from patients treated with atovaquone-proguanil (Malarone) have previously been shown to inhibit the mosquito transmission of Plasmodium falciparum, though the inhibition was not complete and the effect declined 2 weeks after treatment. In marked contrast, the inhibition of transmission of P. berghei by human sera (fed to mosquitoes, with P. berghei gametocytes, via membrane feeders) from volunteers treated with atovaquone-proguanil was total up to day 28 post-treatment and still very significant at day 56. In view of the short half-lives of atovaquone and proguanil, this was unexpected, and further experiments, reported here, were undertaken. In contrast to the incomplete blockade of infectivity of P. falciparum by serum taken 4 days post-treatment, such serum was totally inhibitory against P. berghei at a 1:10,000 dilution, indicating a remarkable sensitivity of P. berghei and demonstrating an unusual difference between the two Plasmodium species in response to a drug. The inhibitory effect on P. berghei after day 4 was caused by atovaquone and mainly through blockade of development from ookinete to oocyst. Despite previous information on the rapid elimination of atovaquone by patients, the present data indicate that low concentrations of this drug may persist in the plasma for some weeks after treatment.

The mosquito transmission of malaria: the effects of atovaquone-proguanil (Malarone) and chloroquine.

Despite its recognized importance, the prevention of patients with malaria from continuing to infect mosquitoes after treatment is not always achieved in practice. An inevitable consequence of the prolonged life-span and relative metabolic stasis of the mature gametocytes of Plasmodium falciparum is that they are not cleared by most antimalarials, and few antimalarials block infection in the mosquito vector. Previous research on the constituents of Malarone, a new 'combined antimalarial', suggested that the active components, atovaquone and proguanil, might inhibit infectivity of gametocytes to mosquitoes. We contrast here the impact of atovaquone-proguanil and chloroquine on the transmission of P. falciparum and P. berghei. While chloroquine enhanced infectivity of P. falciparum, atovaquone-proguanil caused a significant reduction. Surprisingly, sporontocidal activity against the rodent parasite persisted long after the levels of the constituent drugs would have been expected to have fallen below effective plasma concentrations on the basis of the established pharmacokinetics of atovaquone and proguanil. The P. berghei model may thus have provided a sensitive bioassay, detecting drug(s) at levels below that normally found with the usual chemical assays.

The chemotherapy of rodent malaria. LIX. Drug combinations to impede the selection of drug resistance, Part 3: Observations on cyproheptadine, an antihistaminic agent, with chloroquine.

Several compounds in current clinical use as antihistaminic agents, among them cyproheptadine (CYP), have been shown, in experimental models, to reverse resistance of the asexual, intra-erythrocytic stages of rodent or human malarial parasites to chloroquine (CQ). Although preliminary clinical trials with CYP failed to confirm such activity in subjects with naturally acquired infection with Plasmodium falciparum, Nigerian investigators have reported that another antihistaminic, chlorpheniramine, significantly restores the blood schizontocidal action of CQ in semi-immune patients with CQ-resistant P. falciparum, when the two compounds are administered together. The rates at which resistance to CYP can be produced, in mice infected either with CQ-resistant P. yoelii ssp. NS or CQ-resistant P. yoelii nigeriensis, when drug-selection pressure is exerted with this compound alone have now been compared with the rate and extent to which resistance develops in infected animals that are treated with various combinations of CYP and CQ. The data indicate that, in both parasites, stable resistance develops slowly to CYP alone and that exposure to a combination of CYP plus CQ significantly impedes the selection of resistance to CYP. Although the antimalarial action of CYP is reported to extend to the pre-erythrocytic hepatic stages, there was no evidence of gametocytocidal activity in the present study. The future implications of these observations are discussed in relation to the clinical potential of CYP + CQ and similar combinations and possible future research.

The differing impact of chloroquine and pyrimethamine/sulfadoxine upon the infectivity of malaria species to the mosquito vector.

Using serum or infected blood from Danish volunteers and Plasmodium falciparum-infected Mozambican patients, respectively, the impact of curative doses of chloroquine and pyrimethamine/sulfadoxine upon infectivity of P. falciparum to Anopheles arabiensis and An. gambiae or of P. berghei to An. stephensi was studied. Both treatments cleared circulating P. falciparum gametocytes within 28 days. Before this clearance, chloroquine enhanced infectivity to An. arabiensis, whereas pyrimethamine/sulfadoxine decreased infectivity. Patients harboring chloroquine-resistant parasites as opposed to -sensitive ones were 4.4 times more likely to have gametocytes following treatment. In contrast, pyrimethamine/sulfadoxine-resistant parasites were 1.9 times less likely to produce gametocytes. In laboratory infections using replicated P. berghei or P. falciparum preparations, serum from chloroquine-treated, uninfected, nonimmune volunteers enhanced gametocyte infectivity with increasing efficiency for 21 days following treatment, whereas pyrimethamine/sulfadoxine significantly suppressed infectivity. The observed enhancement in infectivity induced by the use of chloroquine combined with increased gametocytemias in chloroquine-resistant strains may in part explain the rapid spread of chloroquine resistance in endemic populations.

Antimalarial drugs and the mosquito transmission of Plasmodium.

It is well-known that whenever possible, the treatment of patients with malaria should include measures to prevent them transmitting the infection to others. This is particularly important for P. falciparum, where the gametocytes can survive for a much longer period than the asexual stages. Not all antimalarials are gametocytocidal or sporontocidal and those that are may have particular disadvantages or lose their effectiveness because of resistance. Even drugs that have no obvious gametocytocidal or sporontocidal activity may have other effects. These include the possibility of increasing transmission, either by affecting the parasite within an individual host or by selection for parasite strains with increased potential for infecting the mosquito vector. This review summarises the available information on the properties of antimalarials in relation to mosquito transmission and highlights the need for more attention to be paid to this aspect of drug action.

Protective responses against skin-dwelling microfilariae of Onchocerca lienalis in severe combined immunodeficient mice.

Inoculation of severe combined immunodeficient (SCID) mice with microfilariae of Onchocerca lienalis results in a sustained infection of the skin, extending for months beyond the point at which the parasites are eliminated from immunocompetent BALB/c controls. Reconstitution of SCID mice with spleen cells, thymocytes, or CD4+-cell-enriched splenocytes from naive BALB/c donors confers the ability to mount a protective immune response, leading to the rapid elimination of microfilariae. High levels of interleukin-5 and low levels of gamma interferon in the sera of reconstituted SCID mice during the destruction of microfilariae suggest that this protective immune response is directed by Th2 lymphocytes, mirroring that observed in immunocompetent controls. Unexpectedly, abbreviation of primary infections of unreconstituted SCID mice with the drug ivermectin induces resistance to reinfection with microfilariae at a level equivalent to that induced in secondarily infected, immunocompetent controls. In contrast to protection mediated by adoptive reconstitution, resistance induced by ivermectin-abbreviated infection occurs in the absence of T cells and in association with negligible levels of serum interleukin-5 and gamma interferon. This points to the activation of some alternative host defense mechanism that operates after the clearance of therapeutic levels of drug. Such a response could have important implications for the treatment of human onchocerciasis and may go some way in explaining the long-term suppression of microfilariae observed in patients after treatment with ivermectin.

Premature removal of uninfected erythrocytes during malarial infection of normal and immunodeficient mice.

Anaemia during blood-stage plasmodial infections is known to be due to at least three mechanisms: direct destruction of erythrocytes by the intra-erythrocytic parasite, inhibition of erythropoiesis, and premature removal of uninfected erythrocytes. The removal of the uninfected erythrocytes is considered by many to be dependent on an antibody-mediated mechanism. Our investigations involving normal, severe combined immunodeficient (SCID) and nude BALB/c mice and the murine malaria parasite Plasmodium yoelii, indicate that this mechanism is unlikely. The process of removal of uninfected erythrocytes was reduced in SCID mice but could not be enhanced by the passive transfer of serum from infected immunocompetent mice. Macrophage activation, as judged by the removal of xenogeneic erythrocytes, did not differ in the three strains of mice. Changes in the erythrocytes themselves may be responsible for their shortened lifespan.

Plasmodium berghei: infectivity of mice to Anopheles stephensi mosquitoes.

The infectivity of P. berghei-infected TO mice to mosquitoes declines rapidly 2 to 5 days after blood inoculation, in spite of rising numbers of gametocytes in the blood. This pattern is typical of many malaria infections and various factors, particularly specific and nonspecific immune responses, have previously been implicated in the decline. Here we report that (1) simple physiological changes in the mouse blood, namely, falling pH and bicarbonate levels induced by high parasitaemias, are responsible for the sustained inhibition of infectivity; (2) the inhibition is reversible in vivo by the addition of sodium bicarbonate alone; (3) the inhibition occurs at the point of exflagellation; (4) contrary to previous observations (Kawamoto et al. 1992), exflagellation in P. berghei, like that in P. gallinaceum (Bishop and McConnachie 1956; Nijhout and Carter 1978; Nijhout 1979) and P. falciparum (Ogwan'g et al. 1993), is dependent on extracellular bicarbonate; and (5) induction of exflagellation by a mosquito factor is bicarbonate dependent. These new observations are critical to the design and interpretation of experiments on other transmission blocking phenomena.

Models for malaria: Nature knows best.

Despite our increasing knowledge of the immunology of malaria in humans and in experimental systems, many questions remain unanswered. Given that this research has not yet led to a successful bloodstage vaccine, perhaps we should look again more carefully, in the light of new knowledge, at how many natural malaria infections are successfully controlled. Of these, the simian malarias are probably the most interesting, as described here by Geoff Butcher.

Nitric oxide synthase activity in malaria-infected mice.

Nitric oxide (NO) is implicated in a variety of major cellular functions including defence from invasion by microbical pathogens. Evidence has been presented suggesting that it is an important mediator of protection in the early non-specific responses to malaria in mice infected with Plasmodium chabaudi (Taylor-Robinson et al. 1993). Other data from in vitro studies on the asexual stages of human parasite Plasmodium falciparum indicated that while nitric oxide itself may not be inhibitory to parasite development, its downstream products do have some anti-plasmodial activity (Rockett et al. 1991) and these could be generated by macrophages (Gyan et al. 1994). Similarly, the sexual phases of both rodent (Motard et al. 1993) and human malaria (Naotunne et al. 1993) are reportedly susceptible to the toxic effects mediated by nitric oxide generated by blood leucocytes in the course of transmission to the mosquito vector.

Malaria and macrophage function in Africans: a possible link with autoimmune disease?

Systemic lupus erythematosus and sarcoidosis, both diseases in which immune responses are aberrant, are not found in Africans in West Africa, but their prevalence in people of West African descent in the USA and UK is higher than that for white people. It is argued here that malaria both prevents these diseases in West Africa by its effects on macrophage function, and has also selected for a predisposition to them.