Presentation of the Plasmodium falciparum antigen Pf155/RESA to human T cells. Variations in responsiveness induced by antigen presenting cells from different but MHC class II identical donors.

The antibody response in humans naturally primed to a malaria vaccine candidate antigen (Pf155/RESA) is genetically regulated. Here, the impact of antigen presenting cells (APC) on the control of in vitro T-cell responses induced by Pf155/RESA or synthetic peptides corresponding to its major Pf155/RESA epitopes was studied. T cells and APC were from the peripheral blood of monozygotic or dizygotic twins and their age matched siblings, all living in the central highlands of Madagascar. When induced to proliferate (thymidine incorporation) in vitro by antigenic peptides, the T-cell responses varied less within the twin pairs than between them and their siblings or the entire group, implying that they were genetically regulated. Occasional MHC class II associations of some of the responses were weak and did not reflect underlying MHC class II restrictions. When T cells and APC from different but MHC class II identical donors were incubated in various combinations, antigen charged APC from homologous donors induced in vitro T-cell proliferation which differed from that induced by the T-cell donors' own APC. Pretreatment of the APC with either paraformaldehyde or anti-class II antibodies inhibited or abolished this antigen dependent T-cell proliferation. The results suggest that the observed differences in T-cell responses induced by APC from different donors reflect differences at the level of these cells. Whether they reflect differences in the proteases involved in antigen processing, in the costimulatory signals provided by the APC to the T cells or in the secretion of other regulatory factors remains to be elucidated.

Longitudinal study of the cellular response to Pf155/RESA and circumsporozoite protein in Madagascar.

In a community follow-up conducted in the Central Highlands of Madagascar, the cellular responses to synthetic peptides from the immunodominant regions of Pf155/RESA and the repeat region of the circumsporozoite protein were studied. Seasonal variations of the T cell response were measured at the individual level; the relationship between this response and the presence of parasites in blood was assessed; the question of the possible protective value of the lymphocyte proliferation in presence of a specific antigen was addressed.

Antibodies to the 4-mer repeat of the ring-infected erythrocyte surface antigen (Pf155/RESA) protect against Plasmodium falciparum malaria.

To investigate the protective role of antibodies to the ring-infected erythrocyte surface antigen (Pf155/RESA) epitopes against Plasmodium falciparum clinical malaria, a cohort study was conducted in a Malagasy village over 7 months. In the 304 individuals included, 127 experienced P. falciparum attacks of under 1500 parasites/microliters with no clinical symptoms (protected individuals) and 177 experienced at least one clinical or preclinical P. falciparum attack requiring therapy (unprotected individuals). Antibodies to whole Pf155/RESA, to single epitopes of the 3' terminus, (EENV)4 and EENVEHDA(EENV)2 had higher responses in protected than in unprotected individuals (P = 0.006, P = 0.005, P = 0.05 respectively). Within the whole pattern of antibodies to the Pf155/RESA epitopes, only anti-R4 was related to protection independently of age and anti-wR. The Pf155/RESA 4-mer repeated epitope might be of interest for inclusion in a vaccine against the asexual blood stages of P. falciparum.

Humoral and cellular immune responses to the circumsporozoite protein of Plasmodium vivax in Madagascar.

Plasmodium vivax malaria is prevalent during the rainy season in the central highlands of Madagascar. In April 1991, we investigated the cellular and antibody immune responses of 53 inhabitants of Manarintsoa, a village in this area, to four antigens corresponding to B and T cell epitopes of the P. vivax circumsporozoite (CS) protein. Cellular responses were assessed by lymphocyte proliferation assay as well as by detection of interferon-gamma and interleukin-2 production in vitro. Cell culture was performed with two overlapping synthetic peptides (CSVTCGVGVRVRSRVNA [amino acids 311-326]) and VRVRSRVNAANKKPED [amino acids 319-334]) from the vicinity of the highly conserved region II of the CS protein. In at least one of the three assays, cells from seven subjects showed a positive response to CSVTCGVGVRVRSRVNA, while cells form 14 subjects responded to VRVRSRVNAANKKPED. Antibodies directed against the two recombinant antigens, NS1(81)V20 and rPvCS-2, both of which contain the entire central repeat region of the P. vivax CS protein, plus regions I and II in the case of rPvCS-2, were measured by the Falcon assay screening test-enzyme-linked immunosorbent assay. Eight and nine subjects had antibodies to NS1(81)V20 and rPvCS-2, respectively. The presence of antibody responses to both recombinant antigens was related (P = 0.02, by Fisher's exact test), but was not related to the presence of a cellular response to peptides from vicinity of region II (P > 0.1, by Fisher's exact test).(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of a nonisotopic DNA assay kit for diagnosing Plasmodium falciparum malaria in Madagascar.

This study evaluated a nonisotopic DNA assay kit for diagnosing Plasmodium falciparum malaria in an area of Madagascar where all Plasmodium species of human malaria are present and where malaria is endemic. Blood samples from 440 healthy children and 20 healthy adults were processed and assayed in a single day in a blind protocol. The parasitemia levels of the four Plasmodium species were determined by microscopic examinations and by counts of numbers of malaria parasites per 1,000 white blood cells. Relative to P. falciparum infections, the DNA assay results agreed with those of microscopy for 207 positive and 239 negative samples; two samples were scored as positive by the DNA probe that were not detected by microscopy, and 12 samples were scored as positive by microscopy but were not detected by the assay. Relative to microscopy, the sensitivity of the assay was 95%, the specificity was 99%, and the effective sensitivity threshold of the DNA probe assay was approximately 30 parasites/mm3 of blood. The assay did not detect infections with either P. vivax, P. malariae, or P. ovale alone, but detected mixed infections of P. falciparum with either P. vivax or P. ovale. With this nonisotopic DNA probe assay, we were able to process large numbers of samples efficiently and to detect P. falciparum malaria infections with high sensitivity and specificity in a population that did not display overt disease symptoms.

Lymphoproliferative responses to synthetic peptides from merozoite ring-infected erythrocyte surface antigen and circumsporozoite protein: a longitudinal study during a falciparum malaria episode.

Proliferative responses of peripheral blood lymphocytes to synthetic peptides representing major epitopes of two malaria antigens (the merozoite ring-infected erythrocyte surface antigen and the sporozoite circumsporozoite protein) were investigated in Madagascar during a clinical Plasmodium falciparum episode. Thirty-seven patients greater than 10 years of age were enrolled at the beginning of the malaria transmission season and followed for four weeks. At enrollment, when the subjects presented with an acute infection, lymphocytes recovered from approximately 30% of them proliferated after peptide stimulation. These proliferative responses decreased sharply one and two weeks after treatment, with less than 10% responding to each peptide. Four weeks after treatment, the responses were only partially restored. The amplitude of these variations was not related to the initial parasitemia. At the individual level, proliferative response to each peptide varied greatly during the followup period, and this variation was unrelated to the presence of parasites in the blood.

Human immune responses to the Plasmodium falciparum ring-infected erythrocyte surface antigen (Pf155/RESA) after a decrease in malaria transmission in Madagascar.

A resurgence of falciparum malaria occurred in the central highlands of Madagascar in the 1980s and was responsible for an outbreak in 1986-1987. Since 1989, transmission has decreased dramatically. In April 1991, we investigated the humoral and cellular immune responses of 53 inhabitants of the village of Manarintsoa to six synthetic peptides that reproduced the major B and/or T cell epitopes of the Pfl 55/ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum. The presence of RESA peptide-reactive T cells was assessed by lymphocyte proliferation assay as well as by detection of in vitro production of interferon-gamma and interleukin-2. The mean values of these cellular responses were low, and the results obtained in these three tests showed no correlation. Twenty-seven subjects presented with anti-RESA antibodies as detected by modified immunofluorescent assay, but the mean levels of anti-peptide antibodies were low. When compared with data obtained in January 1988 from the same subjects with three of the six peptides, the present data demonstrated a decrease in the response to these peptides in terms of both proliferative response and mean antibody titers. The mean values of anti-RESA antibodies remained unchanged. The fact that cellular and humoral responses to the major Pfl 55/RESA epitopes decreased but did not disappear probably reflects both the remainder of the acquired immunity resulting from the 1986-1987 malaria outbreak, and its conservation by the very low level of transmission since 1989.

Antibodies to a Plasmodium falciparum blood-stage antigen as a tool for predicting the protection levels of two malaria-exposed populations.

To evaluate the ability of antibodies to Plasmodium falciparum ring-infected erythrocyte surface antigen (Pf155/RESA) epitopes to discriminate between individuals well protected or poorly protected against malaria, a receiver operating characteristic analysis was performed in two populations living in Madagascar and Malawi. The definition of protection was based on longitudinal measurements of clinical malarial attacks during the season of high malaria transmission in the Madagascar study, and on a cross-sectional measurement of parasitemia in the Malawi study. Antibodies to peptides reproducing the 4-mer, 8-mer, and 11-mer of the Pf155/RESA were tested for their reactivities using the Falcon assay screening test-enzyme-linked immunosorbent assay. Maximal detection of poorly protected individuals (specificity = 100%) corresponded to high cutoff antibody titers (range = 1.65-3.0 optical density [OD] units in the Madagascar study and 0.67-1.42 OD units in the Malawi study) and a sensitivity less than 50%. For a given sensitivity of 50%, specificity ranged from 55% to 62% in the Madagascar study, and from 67% to 94% in the Malawi study. The antibody cutoff titers corresponding to minimal misclassification rates ranged from 0.24 to 1.73 OD units in the Madagascar study and from 0.15 to 0.55 OD units in the Malawi study. For each antibody, the highest detectability value as measured by the area under the curve was obtained for anti-R11 in the Malawi study (0.838). In demonstrating such qualities, antibodies to Pf155/RESA epitopes could be used for screening poorly protected populations in which malaria control programs have to be implemented.

Lack of correlation between HLA class II alleles and immune responses to Pf155/ring-infected erythrocyte surface antigen (RESA) from Plasmodium falciparum in Madagascar.

To investigate the relationships between predominant HLA class II alleles and immune responses to the Plasmodium falciparum ring-infected erythrocyte surface antigen (Pf155/RESA), 50 individuals from the highlands of Madagascar were followed-up from 1988 to 1991. The T cell reactivity and antibody responses to synthetic peptides (EENV)4, (EENVEHDA)4, and (DDEHVEEPTVA)3, representing major T and B epitopes of Pf155/RESA antigen, were assessed with an average of five determinations per individual over the four-year follow-up period. The T cell reactivity was investigated by lymphocyte proliferation and assays for interferon-gamma and interleukin-2 release. Antipeptide antibodies were measured using the Falcon assay screening test-enzyme-linked immunosorbent assay. The cumulative prevalence rates of cellular (range for the three peptides = 64-68%) and antibody responders (range = 70-74%) were similar for each peptide. The HLA class II typing was performed using polymerase chain reaction-restriction fragment length polymorphisms. The prevalent alleles or groups of alleles (frequency > 20%) were similar in responders and nonresponders, both for cellular and antibody responses to each peptide. These were HLA-DR 5 group and HLA-DQA1 *0601, *0101-0102-0104, HLA-DQB1 *0301, and HLA-DPB1 *0101-2601 alleles. Allelic distribution was similar in individuals presenting with (74%) or without (26%) a malaria attack during a 20-week follow-up conducted when malaria was hyperendemic (P > 0.05, by Fisher's exact test). Despite repeated immunologic measures that better identify the responders, no relationship was found between HLA class II alleles and the cellular or antibody responses to Pf155/RESA epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)

Malaria transmission and vector biology in Manarintsoa, high plateaux of Madagascar.

To evaluate the factors which determine the transmission level of falciparum malaria, entomological and parasitological surveys were conducted from October 1988 to February 1990 in Manarintsoa in the central highland plateaux of Madagascar. Mosquitoes were collected for 928 man-nights in pit shelters and indoor resting sites. Malaria vectors were Anopheles arabiensis and An. funestus, with no evidence of the presence of An. gambiae sensu stricto. Vectors were mainly exophilic and zoophilic. The index of stability was less than 1.5. The sporozoite rate was 0.11 for An. gambiae sensu lato and 0.47 for An. funestus. The transmission level was low, with an inoculation rate of 0.91 infective bites/person/year and an infection risk of 0.62. Malaria transmission occurs 7 months of the year in this area, from November to May. Human parasite rates fluctuated from 29% in October to 53% in May.

Humoral and cell-mediated immune responses to the Plasmodium falciparum antigens PF155/RESA and CS protein: seasonal variations in a population recently reexposed to endemic malaria.

Resurgence of falciparum malaria occurred in the Central Highlands of Madagascar in the 1980s and the disease is currently hyperendemic. We determined the humoral and cellular responses to synthetic peptides reproducing the repeat sequences of 2 major Plasmodium falciparum antigens: the Pf155/RESA and the circumsporozoite (CS) protein. Blood samples from 83 subjects living in a rural community near Antananarivo were obtained at the beginning and the end of the transmission season. At enrollment, 40 subjects presenting with and 43 without blood parasites had similar T cell proliferative response and antibody level to all antigens tested. However, P. falciparum-infected individuals exhibited a decrease in the absolute number of T lymphocytes, due to a diminished number of CD8+ and natural killer lymphocytes. The number of CD4+ cells was similar in both groups. In the overall population, 45% of subjects had a T cell response to at least 1 RESA peptide (29-35% responding to a given peptide) and 35% to the CS protein peptide. Thirty-two percent of the donors presented with RESA antibodies and 23% had CS protein antibodies. After 20 weeks, at the end of the transmission season, cellular proliferative responses to all antigens markedly decreased as evidenced by a decrease of both the number of responders and mean stimulation indexes. Humoral response to RESA, as detected by erythrocyte membrane immunofluorescence (number of responders and mean antibody titers) markedly increased. Humoral responses to the CS protein and RESA peptides were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

IgG1 and IgG2 antibody responses to Plasmodium falciparum exoantigens correlate inversely and positively, respectively, to the number of malaria attacks.

In Manarintsoa, near Antananarivo, Madagascar, two groups of patients were defined in terms of malaria clinical immune status: Group MA+ consisted of 36 patients who suffered from between one to four malaria attacks (MA) during the 20-week study, and Group MA- who comprised of 48 persons who did not have any malaria attacks during this time. In group MA+, IgM and IgG antibody levels to Plasmodium falciparum exoantigens (E-Ag) were inversely related to the number of malaria attacks. The level of IgM antibodies were significantly higher in group MA+. In contrast, IgG, IgG1, IgG2, IgG3 and IgG4 antibodies to E-Ag were significantly higher in group MA-. The level of IgG1 antibodies was inversely correlated, and IgG2 antibodies were positively correlated to the number of malaria attacks.

Chronotherapy of malaria: improved efficacy of timed chloroquine treatment of patients with Plasmodium falciparum infections.

The effect of routine treatment with chloroquine (10 mg/kg on days 1 and 2 and 5 mg/kg on day 3) on parasitaemia and parasitaemic profile of patients infected with Plasmodium falciparum was studied. As with P. vinckei petteri, the mid-term trophozoites of P. falciparum were the most susceptible stages to chloroquine treatment. It is suggested that, in order to diminish the frequency of drug administration and to lower the risks of chemoresistance developing, treatment should be diversified, using the drug which is most effective on the parasite stages present in the peripheral blood.

Efficacy of a 3-day oral regimen of a quinine-quinidine-cinchonine association (Quinimax) for treatment of falciparum malaria in Madagascar.

In the search for an effective, safe and field-adapted alternative to chloroquine for therapy of chloroquine-resistant Plasmodium falciparum infections in Africa, a 3-d oral regimen of Quinimax (an association of quinine, quinidine and cinchonine) was evaluated in 35 individuals with P. falciparum in Madagascar, an area with chloroquine resistance. 63% of the parasite strains isolated were resistant in vitro to chloroquine, and 59% of the infections were present despite previous chloroquine intake. Three daily oral doses of 10 mg/kg Quinimax for 3 d cleared parasitaemia and improved clinical status in all subjects. Mean parasite and fever clearance times were 51.7 and 37.4 h, respectively. All patients were aparasitaemic at the end of the 7-d follow-up. When formulating therapy guidelines, the 3-d Quinimax regimen should be considered as a valuable alternative to chloroquine for treating falciparum malaria in African areas with clinical resistance to chloroquine.

Malaria transmission and vector biology on Sainte Marie Island, Madagascar.

A 17-mo longitudinal malaria survey (November 1988-March 1990) was carried out on Sainte Marie Island, an area on the east coast of Madagascar which is frequently visited by tourists. During 706 man-nights of capture, 46,401 mosquitoes belonging to 32 species were caught. Sporozoite rates were determined by ELISA and incriminated Anopheles gambiae Giles s.s., An. funestus Giles, and An. mascarensis De Meillon as vectors of malaria. An. gambiae, the main vector, was highly anthropophilic but largely exophilic. Transmission by this species occurred mainly from November to April; the overall circumsporozoite antigen positivity rate was 1.7%, with a maximum of 3.2% in March-April. The nightly peak of transmission occurred between midnight and 0400 hours. The annual inoculation rate was calculated to be 100 infective bites per human, of which 92 were of Plasmodium falciparum. An. funestus played a minor role in transmission. An. mascarensis, an anopheline endemic to Madagascar, was incriminated for the first time, as a malaria vector. The sporozoite rate in this species varied from 0.4 to 0.9% as shown by both ELISA and salivary gland dissections. Parasite indices in humans up to 20 yr of age fluctuated during the year from 64 to 80%. Bed nets are recommended for malaria protection for the local population and tourists.

Observations ultrastructurales sur les formes sanguines et la sporogonie de plasmodiums de lémuriens malgaches.

The ultrastructure of blood stages and oocysts of Plasmodium coulangesi and P. percygarnhami, both parasites of Madagascan lemurs, was studied. The main results are:

[Ecological factors in the renewed outbreak of malaria in Madagascar]. / Facteurs écologiques de la recrudescence du paludisme à Madagascar.

The new epidemic of malaria which spread on the Madagascar high plateau in 1986-1987 is due to the combination of several factors (some of which are analysed by the authors, especially those related to anopheles, parasite and man). The authors compare the situations on the high Plateau and on St Mary Island, on the East Madagascar Coast, where the malaria is stable. Concerning the vector, the most interesting fact is the come-back of Anopheles funestus on the high Plateau from which it had disappeared at the beginning of the fifties. In this area, An.arabiensis seems to be the only representative of the gambiae complex whereas it is An. gambiae s.s. in St Mary Island. The parasite is getting more and more resistant to chloroquine. Nevertheless, man seems to develop protection, but it is difficult to analyse the markers which would prove the protection. However, that protection was assessed, on the humoral and cellular level, against the peptides of the RESA (Ring Infected Erythrocytes Surface Antigen), the circumsporozoite protein and the antigen E.

[Stability and chemosensitivity of Plasmodium falciparum to chloroquine in 1990 and 1991 in Ankazobe, a village in high plateau Madagascar]. / Stabilité de la chimiosensibilité de Plasmodium falciparum a la chloroquine en 1990 et 1991 a Ankazobe, village des hauts plateaux malgaches.

The in vivo and in vitro response of Plasmodium falciparum to chloroquine was conducted in Ankazobe, a village located in the high plateau area. These studies confirmed the low level of chloroquine-resistance. The in vivo data indicate the absence of increase resistance during the 2 years study. Chloroquine is still the drug of choice for the treatment of malaria attack in this area.

[The impact of curtains impregnated with deltamethrin on the vectors and morbidity of malaria: results in Ankazobe, on the plateaus of Madagascar]. / Impact des rideaux imprégnés de deltaméthrine sur les vecteurs et la morbidité palustre: résultats à Ankazobe, sur les plateaux de Madagascar.

To evaluate the efficacy of deltamethrin impregnated curtains on malaria morbidity in a low transmission area, we studied volunteer families in the village of Ankazobe in the Madagascar Highlands from February 1993 to June 1994. After randomization, we provided 46 houses having 244 inhabitants with impregnated curtains (I) and 45 others having 257 inhabitants with nonimpregnated curtains (NI) as controls. We first estimated the number of mosquito bites in the protected versus nonprotected households. Every month, we captured mosquitos on humans in 6 houses per night for 4 nights. For the I group compared to the NI group, the number of bites by the Anopheles funestus vector per human per night was reduced by 64% in 1993 and 39% in 1994. We also analyzed the malaria morbidity. Malaria morbidity was defined as patients having both temperatures greater than 37.5 degrees C and Plasmodium falciparum parasitemia greater than 1500/microliter with clinical symptoms. From February to July 1993, we observed no significant difference in morbidity: there were 103 cases of malaria among 244 inhabitants of the I group and 117 cases among 257 inhabitants of the NI group. However, during the period of highest transmission from March to May in 1993, there were significantly fewer cases in the I group (68) than in the NI group (94). From January to June 1994, the difference was clear: only 35 malaria cases were observed among the 208 inhabitants of the I group as compared to 65 cases among the 223 inhabitants of the NI group (Chi square = 9.17, p = 0.0024). Inhabitants of the I group could have been contaminated before the curtains were set up. After treatment of the cases and use of curtains during the second year, we observed a reduction in the number of mosquito bites and malaria cases. The small size of the trial made the interpretation of the data difficult. Nonetheless, the results tentatively support the use of impregnated curtains as an antimalaria tool in an integrated control program.

[Drug resistance and therapeutic strategy]. / Chimiorésistance et stratégie thérapeutique.

In Madagascar, Plasmodium falciparum resistance to chloroquine was clinically suspected in 1975 by Goasguen and demonstrated in 1981 by Arronson et al. Since then, many studies were conducted throughout the island, in the North, South, East and West, on the high Plateau and on the coasts. Two methods were used, including an in vivo method similar to the therapeutic standard protocol of the WHO, and an in vitro method employing the semi-microtest of Le Bras and Deloron. From 1982 to 1986, the 291 in vivo tests performed showed that 20% of the strains were of the types R1 or R2 (SR1 included). From 1987 to 1994, of the 621 in vivo tests performed, 369 (59.4%) of the cases responded to treatment. The deterioration of the situation observed in 1988 (Lepers et al.) seemed to be stabilized (Ringwald et al.). No strain of the type R3 was found. In conclusion, we report the absence of strain type R3 and also the clinical efficacy of chloroquine. The action of chloroquine was spectacular on the fevers and there was remarkable reduction of the parasitaemia. Thus, for treating outbreaks of simple malaria in Madagascar, chloroquine remains the best choice if it is administered at an efficacious dose.