Immune pressure selects for Plasmodium falciparum parasites presenting distinct red blood cell surface antigens and inducing strain-specific protection in Saimiri sciureus monkeys.

The passive transfer of specific antibodies to a naive splenectomized Saimiri sciureus monkey infected with the Palo Alto FUP/SP strain of Plasmodium falciparum resulted in the emergence of parasites resistant to the transferred antibodies. Molecular typing indicated that the original and resistant parasites were isogenic. Saimiri monkeys primed with original parasites were fully susceptible to a challenge by the resistant ones, and vice versa. This absence of crossprotection indicates that strain-specific determinants would be the major targets of protective immunity developed in these monkeys. Phenotypic analysis showed that the surface of the infected red blood cells differed in both lines. Original parasites formed rosettes, autoagglutinated, presented characteristic knobs at the surface of the infected red blood cell, and did not agglutinate in the presence of a pool of human immune sera. In contrast, the resistant parasites did not form rosettes, did not spontaneously autoagglutinate, presented abnormal flattened knobs, and formed large aggregates in the presence of a pool of human immune sera. The presence of strain-specific determinants at the surface of the resistant parasites was confirmed by surface immunofluorescence and agglutination using homologous Saimiri serum. Neither the original nor the resistant parasites cytoadhered to an amelanotic melanoma cell line, suggesting that cytoadherence and agglutination can be dissociated. These results indicate that parasites that differ by the antigens exposed at the surface of the red blood cell induce strain-specific immunity. Furthermore they show that rosetting and nonrosetting parasites differ in their antigenic properties and do not crossprotect.

Ascites production in the squirrel monkey (Saimiri sciureus).

A method is described for inducing the production of large amounts of ascitic fluid (AF) in the squirrel monkey Saimiri sciureus. The total amount of protein in the induced AF is close to 60% of that in the serum. Electrophoretic analysis of serum and AF samples from the same monkey revealed similar protein patterns, including gamma globulins. Antibody titers against Plasmodium falciparum in infected monkeys, measured by indirect immunofluorescence, were also comparable in serum and AF.

A two-site sandwich immunoradiometric assay of squirrel monkey (Saimiri sciureus) IgM using monoclonal antibodies.

Nine hybrid clones secreting antibodies to squirrel monkey (Saimiri sciureus) IgM were produced and two of these (1F1G5 and 5H11B3) were selected for further studies. These non-precipitating monoclonal antibodies reacted with two distinct repetitive antigenic determinants, probably of the conformational type, only present on the native or SDS-denatured IgM molecule. Reduction of the pentamer with 2-mercaptoethanol led to complete destruction of the corresponding epitopes. 1F1G5 antibodies from ascitic fluids were used in the purification of monkey IgM by affinity chromatography. The characteristics of 1F1G5 and 5H11B3 MAbs permitted the development of a solid-phase two-site immunoradiometric assay for the measurement of IgM levels in serum specimens taken from healthy animal donors of both sexes.

The virulent Saimiri-adapted Palo Alto strain of Plasmodium falciparum does not express the ring-infected erythrocyte surface antigen.

The Palo Alto strain of Plasmodium falciparum is highly virulent for the Saimiri sciureus monkey. We have observed that these parasites do not express the Ring-infected erythrocyte surface antigene (RESA) gene. Immunoblots indicated that the Pf155/RESA protein was absent. The RESA mRNA could not be detected. Polymerase chain reaction and Southern blot analysis demonstrated that this lack of expression is due to gene rearrangements. The majority of the Palo Alto parasites have a deletion of the entire RESA gene, whereas in a minor fraction the RESA sequences remain detectable, but the 5' miniexon 1 is inverted. These data show that the RESA protein is dispensable for in vivo parasite growth, at least in Saimiri monkeys.

In vivo and in vitro derived Palo Alto lines of Plasmodium falciparum are genetically unrelated.

The Uganda Palo Alto strain of Plasmodium falciparum (FUP) is routinely used as a reference isolate in a number of laboratories. It is one of the few P. falciparum strains that can both be propagated in vivo in monkeys and maintained in culture. The Palo Alto parasites have been characterized for several biochemical and molecular markers, but many of the data reported so far are contradictory. We have analyzed and compared by Southern blotting, PCR and DNA sequencing, several DNA preparations obtained from different FUP lines and from the FCR3 strain. We show here that FUP lines propagated in Saimiri monkeys (FUP/S) and those maintained in culture (FUP/C) for many years in our laboratory differ in the various genetic markers investigated (P190, MSA2, S-Ag, KAHRP, 96 tR, pPFPA rep 20 and pPF 11.1). Therefore, at the present, two genetically unrelated strains of P. falciparum widely distributed over numerous laboratories are designated FUP/Palo Alto. When the Saimiri-propagated FUP/S line was used to initiate an in vitro culture in human red blood cells, no evidence of instability or genetic drift was obtained. The growth rate and genomic characteristics remained constant for several months. Likewise, the FUP/C line was found unchanged after three transfers in Saimiri monkeys. FUP/CP parasites were shown to be genetically closely related to FCR3. In addition, a subline of FUP/C strain selected by repeated flotation on gelatin was found to differ in several characters such as KHARP, P190 and S-antigen genes, which are known to be located on different chromosomes.

A large multigene family expressed during the erythrocytic schizogony of Plasmodium falciparum.

We report the identification of a large multigene family of Plasmodium falciparum using a clone isolated with a polyclonal antiserum raised to a Babesia divergens merozoite protein. The recombinant antigen reacted with human sera collected from individuals exposed to malaria. The deduced protein sequence contains a motif homologous to the consensus sequence of merozoite rhoptry proteins encoded by multigene families in several Babesia species. Antibodies raised to the recombinant protein reacted with a 60-kDa merozoite protein both on B. divergens and on P. falciparum immunoblots. The insert hybridized to a large number of fragments on P. falciparum Southern blots and to most chromosomes of the parasite. Specifically, approx. 3-kb RNAs were detected in 4-16-nucleus schizonts. Ten distinct cDNAs were isolated that differed in the size, position and number of restriction sites in the region homologous to the original genomic clone. With about 140 copies per haploid genome, this is the first large multigene family described in malaria parasites. The existence of a multigene family encoding proteins present in the invasive stage of malaria parasites suggests an important role in invasion and denotes a significant potential for generating diversity.

Variant- and strain-specific immunity in Saimiri infected with Plasmodium falciparum.

Variant- and strain-specific immunity to malaria in Saimiri monkeys infected with homologous O and R variants of the Palo Alto strain (FUPSP) of Plasmodium falciparum or by various heterologous divergent strains were studied. Following homologous reinfections, the primary immune response in monkeys was effective only against the same variant type but not against the other variant, which differed only by antigens exposed at the surface of the infected red blood cell. In contrast, after two successive inoculations with a single variant type, a variant transcending immunity developed to both O and R parasite populations. The immunity against FUPSP in monkeys repeatedly infected with various combinations of heterologous strains, including Sal I, Tanzania, Camp, FUPCP, FCH4, FVO, and FUPCDC parasites was less effective, resulting at best in protecting the monkey against fulminating infection. However, in several cases, previous or concomitant heterologous infections modified the course of virulent infection by FUPSP parasites, indicating a significant degree of cross-protection between the strains. Therefore, in this model, while variant- and strain-specific antigens are important components of acquired immunity to malaria, the monkey immune response to infection transcends phenotypic antigenic variation and strain diversity.

Saimiri sciureus (karyotype 14-7): an alternative experimental model of Plasmodium falciparum infection.

In an earlier manuscript, we described the first phase of adaptation of the Palo Alto I strain of Plasmodium falciparum to Saimiri sciureus (squirrel monkeys). Now, after more than 50 P. falciparum blood transfers in splenectomized Saimiri, the parasite has become fully adapted to this experimental host. A highly reproducible pattern of infection is evident in these splenectomized animals, which is characterized by a rapidly rising parasitemia and a lethal outcome. In intact animals, the course of infection is extremely variable, with a tendency towards high parasitemias and low survival rates. After 60 passages, the parasites have maintained their invasiveness for human red blood cells and can easily be propagated by continuous in vitro culture. Conversely, prolonged in vitro culture of this parasite strain has not decreased its infectivity for Saimiri. Intact, P. falciparum-infected animals rapidly develop a high degree of resistance to reinfection, even when treatment is initiated shortly after detection of the first circulating parasites. Under identical conditions, splenectomized animals develop a variable degree of protection. The potential usefulness of squirrel monkeys as experimental hosts of P. falciparum and P. vivax malaria is discussed in light of the present findings.

Different genetic characteristics of Plasmodium falciparum isolates collected during successive clinical malaria episodes in Senegalese children.

A narrow epidemiologic survey was conducted during a four-month period of intense malaria transmission in Dielmo, a holoendemic Senegalese village. Longitudinal clinical and parasitologic follow-up indicate that clinical malaria episodes always occurred after an abrupt increase in parasite densities. Polymerase chain reaction analysis of Plasmodium falciparum parasites was carried out in blood samples collected longitudinally from 10 children who had experienced several clinical episodes during this period. Our data show that the genetic diversity of the parasites circulating in this village is very large. The successive clinical episodes experienced by each child were caused by genetically distinct parasite populations that were recently inoculated and multiplied in an apparently unrestricted manner. Importantly, the genetic characteristics of the parasite populations detected during phases of asymptomatic carriage differed from those causing a clinical episode, suggesting that the various factors that control of parasite growth in these children are strain-specific.

Rapid turnover of Plasmodium falciparum populations in asymptomatic individuals living in a high transmission area.

A polymerase chain reaction (PCR) typing technique, based on the amplification of polymorphic regions from the merozoite surface protein 1 (MSP-1) and MSP-2 Plasmodium falciparum genes, was used to characterize parasites collected in a longitudinal study of asymptomatic carriers of malaria parasites living in two distinct epidemiologic situations. Blood samples were collected from children and adults living in the village of Dielmo, Senegal, when malaria transmission was 3-6 infective bites/week/individual. For each individual, every sample collected at two-week intervals over a period of three months showed a specific PCR pattern. Changes involved both appearance and disappearance of specific alleles. Analysis of blood samples collected at a few-days interval showed that modifications of the PCR patterns occurred rapidly. Most alleles were detected over a period of 2-3 weeks, but some alleles could be detected only for a few days. The frequent modifications of the PCR patterns indicate significant changes in allelic balance over time, and importantly, this was observed both in children and adults. These results strongly contrast with the stability of the parasite types harbored by asymptomatic individuals living in Pikine, Senegal during a period in which malaria transmission was interrupted, and therefore suggest that the rapid turnover observed in Dielmo may reflect the introduction of new parasite populations by mosquitoes.

Plasmodium falciparum parasites in French Guiana: limited genetic diversity and high selfing rate.

The genetic characteristics of Plasmodium falciparum isolates collected in French Guiana, where malaria transmission is low and occurs in isolated foci, were studied. Blood samples were collected from 142 patients with symptomatic malaria and typed using a polymerase chain reaction-based strategy for merozoite surface protein-(MSP-1) block 2, the MSP-2 central domain, and glutamate-rich protein (GLURP) repeat domain polymorphism. This showed that the parasite population circulating in French Guiana presented a limited number of allelic forms (4, 2, and 3 for MSP-1 block 2, MSP-1, and GLURP, respectively) and a small number of mixed infections, contrasting with the large genetic diversity of parasite populations and infection complexity reported for Africa, Asia, and other parts of South America. Two groups of isolates displaying identical 3 loci allele combinations were further studied for the Pf332 antigen, histidine-rich protein-1, thrombospondin-related anonymous protein, and Pf60 multigene family polymorphism. Within each group, most isolates were identical for all markers tested. This suggests a high rate of self-fertilization of P. falciparum parasites in French Guiana, resulting in homogenization of the population. The implications of these findings for malaria control in areas of low endemicity are discussed.

Extensive genetic diversity of Plasmodium falciparum isolates collected from patients with severe malaria in Dakar, Senegal.

While some genetic host factors are known to protect against severe Plasmodium falciparum malaria, little is known about parasite virulence factors. We have compared the genetic characteristics of P. falciparum isolates collected from 56 severe malaria patients and from 30 mild malaria patients recruited in Hôpital Principal, Dakar, Senegal. All isolates were typed using polymerase chain reaction amplification of polymorphic genetic loci (MSP-1, MSP-2, HRP1, GLURP, CSP, RESA, and the multigene family Pf60). The complexity of infections was lower in severe than in mild malaria and the parasite genetic diversity in both groups was very large. No specific genetic make-up was associated with severity; there were, however, marked differences in allele frequencies in both groups, with a prevalence up to 60% of MSP-2 alleles specifically observed in the severe malaria isolates. In addition, the presence of MSP-1/RO33 alleles was significantly associated with a higher plasma level of tumour necrosis factor alpha receptor 1 (P < 0.05), a reported indicator of severity in human malaria. These results point to potential differences in the genetic characteristics of parasites inducing severe versus mild pathology.

A study of the genomic diversity of Plasmodium falciparum in Senegal. 2. Typing by the use of the polymerase chain reaction.

The genomic polymorphism of Plasmodium falciparum was investigated in a series of samples collected in Senegal during one transmission season. PCR analysis was performed on several genes coding for blood-stage antigens: the gene for the major merozoite surface antigen P190, the gene for the second merozoite surface antigen MSA2 and the gene coding for antigen 96tR/GBP130. In each case, several distinct forms of the genes studied were observed. Both the MAD20 and K1 allelic families of P190 genes were observed. PCR analysis of a single variable region did not differentiate each isolate. However, when the data obtained for several markers are combined, each isolate had a specific genotype. Thus, using PCR to study in parallel several loci is a useful tool to genetically type strains.

[Subsidized artemisinin based combination treatments for Africa]. / Associations à base d'artémisinine subventionnées pour l'Afrique.

Malaria-associated mortality and morbidity have increased in recent decades, with the worldwide spread of chloroquine and sulfadoxine-pyrimethamine resistant parasites. Artemisinin-based combination therapies (ACTs) have been proposed as an alternative to conventional antimalarial drugs. ACTs are effective against multidrug-resistant infections, work quickly, are safe and well tolerated, and seem to decrease transmission by inactivating gametocytes. The affordable medicines facility-malaria (AMFm) - an initiative aiming at increasing the availability of affordable ACTs through public and private practice - is trying to accelerate the large-scale use of ACT worldwide. This began with an initial pilot phase in a selected group of African countries. However, the epidemiology of malaria, the economic context, and healthcare infrastructure of African countries differ considerably from those prevailing in Asia, where ACTs were first implemented in the 1990s. ACT implementation in Africa must therefore be accompanied by control and operational measures to maintain the efficacy of ACT and to protect patients against misuse. We discuss the expected benefits of the AMFm initiative in Africa and stress the importance of dealing with operational issues before implementation in the field, focusing particularly on drug resistance.

A complement receptor-1 polymorphism with high frequency in malaria endemic regions of Asia but not Africa.

Complement receptor-1 (CR1) is a ligand for rosette formation, a phenomenon associated with cerebral malaria (CM). Binding is dependent on erythrocyte CR1 copy number. In Caucasians, low CR1 expressors have two linked mutations. We determined the Q981H and HindIII RFLP distribution in differing population groups to ascertain a possible role in adaptive evolution. We examined 194 Caucasians, 180 Choctaw Indians, 93 Chinese-Taiwanese, 304 Cambodians, 89 Papua New Guineans (PNG) and 366 Africans. PCR/RFLP used HindIII for CR1 expression and BstNI for the Q981H mutation. DNA sequencing and pyrosequencing were performed to resolve inconclusive results. Gene frequencies for the L allele were 0.15 in Africans, 0.16 in Choctaws, 0.18 in Caucasians, 0.29 in Chinese-Taiwanese, 0.47 in Cambodians and 0.58 in PNG. Allelic frequency for 981H were 0.07 in Africans, 0.15 in Caucasians, 0.18 in Choctaws, 0.29 in Chinese-Taiwanese, 0.47 in Cambodians and 0.54 in PNG. The Q981H polymorphism correlates with the HindIII RFLP in most groups except West Africans and appears to be part of a low CR1 expression haplotype. The gene frequency for the haplotype is highest in the malaria-endemic areas of Asia, suggesting that this haplotype may have evolved because it protects from rosetting and CM.

Plasmodium falciparum: genetic stability of the Uganda Palo Alto strain propagated in the squirrel monkey (Saimiri sciureus).

The Uganda Palo Alto strain of Plasmodium falciparum has been extensively used in several laboratories to infect Saimiri monkeys. In the study reported here three distinct lines derived by parallel serial blood transfers from a single 10-year-old passage of the Palo Alto strain were examined for genetic diversity using the polymerase chain reaction and restriction mapping techniques. A comparison of the PF 11.1, P190, MSA2, S-Ag, and KAHRP genes indicates that these parasite lines are apparently homogeneous and stable. Nevertheless, microheterogeneity was observed with molecular probes which are known to easily detect restriction fragment length polymorphisms (rep 20 and telomeric probes). These data show that the genetic characters of the strain can be considered monomorphic and are conserved after multiple passages in the squirrel monkey.

In vitro and in vivo assessement of the antimalarial activity of sergeolide.

The antimalarial activity of sergeolide (a quassinoid from PICROLEMMA PSEUDOCOFFEA) was investigated both, IN VITRO on PLASMODIUM FALCIPARUM cultures and IN VIVO through a classical test of schizontocidal action against PLASMODIUM BERGHEI in mice. Sergeolide showed a very strong antiplasmodial activity IN VITRO as well as IN VIVO. Low concentrations (0.006 microg/ml) were able to fully inhibit the IN VITRO growth of chloroquine-sensitive and resistant strains of P. FALCIPARUM. Small amounts (0.26 mg/kg/day) markedly reduced the virulence of experimentally induced P. BERGHEI infection in mice. However, sergeolide, because of its high toxicity (LD 50: 1.8 mg/kg), does not seem, in its present form to be useful for malaria curative treatment.

Study of some parameters affecting the in vitro cultivation of Plasmodium falciparum within Saïmiri sciureus red blood cells.

The in vitro growth and multiplication of the erythrocytic stages of Plasmodium falciparum within Saïmiri sciureus (squirrel monkey) red blood cells have been studied. Various parameters, such as the origin of the red blood cells and serum supplement, nature of the buffer, influence of the final pH of the medium, role of proteose peptone and glucose addition, were investigated. The selection of the best culture conditions led to the obtention of a reproducible in vitro growth of two parasite cycles in Saïmiri erythrocytes, which is an useful achievement for in vitro studies. Our failure to establish a continuous culture line for longer than 19 days, could be explained by a dramatic increasing of osmotic fragility of the Saïmiri red blood cells related to their small size.

[ELISA test in sero-epidemiologic studies of human malaria. Evaluation of an antigen prepared from erythrocytes of Saimiri sciureus experimentally infected with Plasmodium falciparum]. / Le test ELISA dans les études séro-épidémiologiques du paludisme humain. Evaluation d'un antigène préparé à partir des hématies de Saimiri sciureus expérimentalement infectés par Plasmodium falciparum.

A soluble antigen was prepared from squirrel monkeys (Saimiri sciureus) infected with blood-passaged Plasmodium falciparum, and evaluated in an enzyme-linked immunosorbent assay (ELISA) as a potent antigen for malaria diagnostic. The specificity, reproducibility and sensibility of the test showed the suitability of our antigenic preparation in detecting antibodies, IgG and IgM, in patients with P. falciparum infections. Thus, the Saimiri monkey represents an interesting alternative to the classicals sources of parasites from which P. falciparum antigens are commonly extracted (continuous culture and natural infections).

Characterization by anti-Ig monoclonal antibodies of protective and non-protective antibodies against asexual forms of Plasmodium falciparum in the Saimiri monkey.

Four monoclonal antibodies (mAb) which recognize distinct epitopes on Saimiri immunoglobulins were successfully used to characterize the protective and non-protective antibodies developed by this experimental host in response to infection by the human malaria parasite Plasmodium falciparum. Two of these mAb, 3F11/G10 and 3E4/H8, were IgG-specific and directed against conformationally conserved epitopes on the intact molecule. mAb 3A2/G6 and 4G3/B5 were specific for epitopes on two distinct L chain types of all Ig. Radioimmunoassay and immunoblots indicated that L-chains defined by 3A2/G6 were present in IgG molecules of type 3F11/G10, while L chains defined by 4G3/B5 were associated with IgG of type 3E4/H8. The use of these four mAb as immunoadsorbents allowed the purification of two IgG and two Ig populations. When assayed in vivo by passive transfer experiments in recipient monkeys previously infected by P. falciparum, protection could be associated with the IgG or Ig populations of type 3F11/G10-3A2/G6. In contrast, recipient animals which received the IgG type 3E4/H8 or Ig type 4G3/B5 presented rapidly evolving parasitemia similar to that in animals which received non-immune IgG or protective immune sera depleted of the above Ig or IgG fractions. The identification of protective and non-protective Ig populations will help in the evaluation of vaccine candidates against P. falciparum.