R. S. Phillips

Current Status of Malaria and Potential for Control

SUMMARY Malaria remains one of the worlds worst health problems with 1.5 to 2.7 million deaths annually; these deaths are primarily among children under 5 years of age and pregnant women in sub-Saharan Africa. Of significance, more people are dying from malaria today than 30 years ago. This review considers the factors which have contributed to this gloomy picture, including those which relate to the vector, the female anopheline mosquito; to human activity such as creating new mosquito breeding sites, the impact of increased numbers of people, and how their migratory behavior can increase the incidence and spread of malaria; and the problems of drug resistance by the parasites to almost all currently available antimalarial drugs. In a selective manner, this review describes what is being done to ameliorate this situation both in terms of applying existing methods in a useful or even crucial role in control and prevention and in terms of new additions to the antimalarial armory that are being developed. Topics covered include biological control of mosquitoes, the use of insecticide-impregnated bed nets, transgenic mosquitoes manipulated for resistance to malaria parasites, old and new antimalarial drugs, drug resistance and how best to maintain the useful life of antimalarials, immunity to malaria and the search for antimalarial vaccines, and the malaria genome project and the potential benefits to accrue from it.

Plasmodium chabaudi: Antigenic variation during recrudescent parasitaemias in mice

Abstract The A/S strain of Plasmodium chabaudi at different times was twice mosquito passaged and cloned by limiting dilution. Large groups of NIH mice were infected with 10 5 parasitized red cells of populations of parasites which were considered to be identical or very similar to the population forming the first erythrocytic parasitaemia seen in mice after mosquito transmission of the parasite. Most of the mice were killed immediately after the first patent parasitaemia had become subpatent and their sera pooled. The parasitaemias of surviving mice were followed until recrudescences appeared. The protective activity of the immune serum was then tested against the original infecting population and recrudescent populations by passive transfer tests in naive mice. Protection was measured as a delay in patent parasitaemia reaching 2% compared with normal serum recipients. The immune serum significantly delayed the 2% parasitaemia but in different experiments six out of seven recrudescent populations were found to be less sensitive to the effects of the immune serum than the original infecting population. The recrudescent populations retained their reduced or total insensitivity to the action of the immune serum after two blood passages and after eryopreservation. It appears, therefore, that P. chabaudi can undergo antigenic variation.

Immunity to malaria. II. Serological response of monkeys sensitized by drug-suppressed infection or by dead parasitized cells in Freund's complete adjuvant.

Abstract Sera from monkeys sensitized with drug-suppressed infections contained intrastrain variant-specific agglutinins and opsonins for schizontinfected cells, and partially inhibited parasite multiplication in vitro; no evidence of lysins for 51Cr-labeled schizont-infected cells was obtained. Some sera contained precipitins to parasite antigens. Strain differences could be detected by the schizont-infected cell agglutination (SICA) and precipitin techniques. Prechallenge sera from Freunds incomplete adjuvant (FIA)- and Freunds complete adjuvant (FCA)-sensitized monkeys contained high levels of variant specific agglutinins for schizont-infected cells. Sera from FCA-sensitized animals contained opsonins, and these sera were also more reactive in precipitin tests than sera from FIA-sensitized or chronically infected monkeys. SICA tests on parasites isolated from FIA- and FCA-sensitized monkeys after challenge with the homologous variant showed that the breakthrough parasites were of a different intrastrain variant from that used to sensitize and challenge. After challenge with a heterologous variant, no such change in the parasite population was apparent. About half the monkeys immunized with parasitized cells in FCA eradicated the challenge infection after a brief parasitemia culminating in the appearance of abnormal parasites in the blood. Post-challenge sera partially inhibited parasite multiplication in vitro, but lysins specific for schizont-infected cells could not be detected. Abnormal “crisis” forms observed in vivo were not seen in vitro, and the tests used did not explain the unusual sterilizing immunity.

Plasmodium berghei: passive transfer of immunity by antisera and cells.

Abstract Protection against homologous challenge with Plasmodium berghei was transferred from recovered to normal inbred August rats with serum but more effectively with cells. Spleen cells, peritoneal exudate, and a mixture of lymph node, thymus, and bone marrow cells were all effective. Cells were effective after inoculation either intravenously or intraperitoneally. Protection was measured by comparing the height and duration of the primary parasitemia and the duration of the chronic infection following the primary parasitemia, in normal and immune cell recipients. Immune donors were used when all or most had eliminated the parasites unaided. Transfer of immune spleen and lymph node cells between rats of the same sex with a donor-recipient ratio of 1:1 resulted in the recipients effecting a complete cure within 13–20 days of infection, infection being made 5 hr following cell transfer. When challenge was delayed to 69 days after transfer, the parasite was again eliminated by day 16. A reduction in the immune donor to recipient ratio to 1:2 prevented the recipient effecting a rapid cure although the patent parasitemia was shortened. If the transfer was made from male to female rats the donor cells were apparently rejected, and in these circumstances the immunity conferred became less effective with delay in challenging after the transfer. Serum from some rats was nonprotective while their cells were protective. Disintegration of immune cells destroyed their ability to protect. Prior splenectomy of the recipient rats in one experiment prevented them from eliminating the parasite when the majority of their intact counterparts did so. Serum from recipients of immune cells taken on day 13 after infection when their blood was noninfective to mice had the highest activity in a passive protection test.

Antigenic variation during malaria infection—the contribution from the murine parasite Plasmodium chabaudi

P. chabaudi AS strain in laboratory mice provides an accessible and useful model for investigating antigenic variation in malaria parasites. Evidence that P. chabaudi AS undergoes antigenic variation is summarized. A live indirect fluorescent test (IFAT) detects a variable antigen on the surface of schizont‐infected erythrocytes. Five different variable antigen types (VATS) (detected using the live IFAT) are described from a cloned mosquito transmitted parent population. Even during the rising primary parasitaemia VATS switch at high and variable rates (1.2–1.6%). Work towards cloning genes coding for the variable antigen is briefly summarized. Acquired immunity to blood‐stage P. chabaudi AS is initially mediated through Th1 CD4+ T cells and after the primary parasitaemia there is a switch to Th2 CD4+ T cells. Acquired immune effector mechanisms are discussed in the context of antigenic variation by the parasite.

Malaria parasites undergo antigenic variation at high rates in vivo

The rates of switching between expression of variable antigen types (VATS) have been investigated in vivo in a cloned line of Plasmodium chabaudi chabaudi. VAT-specific hyperimmune sera combined with an immunogold-silver staining technique were used to detect VATS, and five estimates of VAT-specific switching rates were determined for three of them. VAT-specific switching rates were consistent for each VAT and differed between VATS in the range 1.3 x 10-2 to 4.3 x 10-4 switches per schizont per generation. This variation suggests that hierarchical expression of VATS in an infection may be determined by switch rates. A minimum estimate of the overall switching rate was determined by summation of the VAT-specific rates in each of two experiments. In both cases the results showed that at least 1 in every 80 schizonts switched VAT expression every generation. This is the first report of antigenic switching rates for malaria parasites measured in vivo in the presence of minimal specific immune pressure, and is the first to show that VAT-specific switching rates vary between VATS. We conclude that switching is rapid and spontaneous, and is regulated, at least in part, by the VATS involved.

Culture of Plasmodium falciparum in vitro: a subculture technique used for demonstrating antiplasmodial activity in serum from some Gambians, resident in an endemic malarious area.

A technique is described for obtaining consistent reinvasion and multiplication of the erythrocytic stages of Plasmodium falciparum from humans in vitro . Subsequent to this, P. falciparum was subcultured through almost three cycles in vitro , during which multiplication of the parasite was obtained in the first but maintenance or only slight increase in numbers was obtained in the second subculture. The effect of sera from adult Gambians on the reinvasion of P. falciparum was measured in vitro both morphologically and by the incorporation of [ 14 C]isoleucine into parasite protein and by the incorporation of [ 3 H]adenosine into parasite DNA and RNA. Two sera reduced the amount of reinvasion.

Immunity to malaria: III. Possible occurrence of a cell-mediated immunity to Plasmodium knowlesi in chronically infected and Freund's complete adjuvant-sensitized monkeys

Abstract Many of the monkeys sensitized with schizont-infected cells in Freunds complete adjuvant (FCA) survived challenge with homologous Plasmodium knowlesi . In view of the ability of FCA to promote induction of cell-mediated immunity, evidence was sought of delayed hypersensitivity and lymphocyte sensitization in monkeys which survived infection. The results of three groups of experiments are presented: (1) skin tests by intradermal injection of parasitized erythrocytes into chronically infected and FCA-immunized monkeys; (2) in vitro thymidine uptake (as a measure of DNA synthesis) by spleen and lymph node cells from sensitized and sensitized and challenged monkeys in the presence of specific and nonspecific antigens; and (3) measuring the release of 51 Cr label from parasitized cells in the presence of immune and normal spleen cells. Examination of the intradermal injection sites revealed immediate reactions in both chronically infected monkeys and in monkeys sensitized with schizont-infected cells in Freunds adjuvant; in the former the immediate reaction increased with the duration of the infection. In the FCA-sensitized monkeys there was evidence of a delayed-type response. Both spleen and lymph node cells from FCA-sensitized monkeys were responsive in terms of increased DNA synthesis in vitro to the sensitizing antigen. This correlates with delayed hypersensitivity. No specific cytotoxic activity of immune spleen cells from FCA-sensitized or chronically infected monkeys on parasitized cells could be detected. A model system using sheep red cells gave positive results. Although these results suggest that in the FCA-sensitized monkeys there is a delayed hypersensitivity to malarial antigens, we do not know if this is involved in the protective mechanisms associated with sensitization with schizontinfected cells in Freunds complete adjuvant.

Parasite-specific IgM plays a significant role in the protective immune response to asexual erythrocytic stage Plasmodium chabaudi AS infection

A comparison of Plasmodium chabaudi AS infection in BALB/c and BALB/c IgM‐deficient mice demonstrated a protective role for IgM during infection. IgM−/– mice, unlike µMT mice, display competent B cell humoral immune responses. Increased susceptibility of IgM−/– mice was demonstrated by increased mortality, an advanced ascending infection and higher peak parasitaemia, as well as enhanced anaemia and weight loss compared with wild‐type mice. The recrudescent parasitaemias were also higher in the IgM−/– mice. Early specific IgM production in P. chabaudi‐infected wild‐type mice was followed by IgG1 and IgG2a production, while IgG1 and IgG2a production in IgM−/– mice was preceded by specific IgD production. No protective role for natural IgM against P. chabaudi AS infection was detected as passive transfer of naïve WT serum into IgM−/– mice did not alter the disease outcome or reduce parasite numbers. Passive transfer of WT antiserum, containing predominantly specific IgM, into IgM−/– mice delayed the ascending parasitaemia and reduced mortality. Similarly, coating parasitized red blood cells with WT antiserum, but not IgM−/– antisera, prior to infection also slightly delayed the ascending acute parasitaemia. Specific IgM therefore plays an important role in the limitation of parasite replication during asexual erythrocytic P. chabaudi AS infection.

Antigenic variation in Plasmodium chabaudi: analysis of parent and variant populations by cloning.

Summary Nineteen of 22 recrudescent populations of Plasmodium chabaudi AS strain were found to be significantly less sensitive to the protective activity of pools of immune serum, than the parent population from which they were derived. The immune sera were collected from donor mice which had been infected with the parent population and had just reduced the patent primary parasitaemia to subpatent levels. Clones prepared from the parent population (which had previously been cloned) and recrudescent variant populations were tested for their sensitivity to the immune sera. It was found that all the clones from the parent population were sensitive to the immune sera but some were more sensitive than others and that a recrudescent variant population could include both sensitive and insensitive parasites. Two insensitive clones of the recrudescent population were found to be different from each other