Malaria vaccines 1985-2005: a full circle?

Few who were actively engaged in malaria vaccine research 20 years ago (including myself) would have imagined that, in 2005, there would still be a prediction of a 10-20-year horizon before vaccines become part of malaria-control strategies. Why is it still proving so challenging to produce effective vaccines

Sympatric Plasmodium falciparum isolates from Venezuela have structured var gene repertoires

BACKGROUND: The human malaria parasite Plasmodium falciparum expresses adhesins belonging to the erythrocyte membrane protein 1 (PfEMP1) family on the surface of the infected host erythrocyte. These antigens elicit a strain-specific antibody response that is associated with protection from disease. During clonal expansion of blood-stage parasites, the surface phenotype of the infected erythrocyte changes because of transcriptional switching among the 40 to 50 members of the highly polymorphic var multi-gene family which encode PfEMP1 variants. Studies to date have compared var repertoires of natural isolates from various geographical locations but have not addressed any within-population structure that may exist among repertoires. METHODS: Distinct parasite genotypes from a single population co-circulating among a defined group of hosts were selected. PCR products encoding the DBL-α domain of PfEMP-1 were cloned and sequenced from each of three isolates. Repertoire similarity was statistically evaluated using combinatorial analysis. The chromosomal location of shared sequences was inferred from similarity to dbl-α of known location in the 3D7 genome. RESULTS: Sympatric parasites were found to share few var gene sequences, even when alleles at other polymorphic loci were shared. A number of the sequences shared by at least two of the isolates studied were found to be related to 3D7 genomic sequences with non-telomeric chromosomal locations, or atypical domain structures, which may represent globally conserved loci. CONCLUSION: The parasite population studied is structured, with minimal overlap in PfEMP1 repertoires. The var gene family accumulates diversity more rapidly than other antigen genes examined. This may be facilitated by ectopic recombination among the sub-telomeric regions of P. falciparum chromosomes

The addition of artesunate to chloroquine for treatment of Plasmodium falciparum malaria in Gambian children delays, but does not prevent treatment failure.

In a randomized controlled trial, chloroquine monotherapy was compared with the combination of artesunate and chloroquine for treating uncomplicated Plasmodium falciparum malaria in 536 Gambian children. Chloroquine-treated children exhibited a 28-day clinical failure rate of 15% (95% confidence interval [CI] = 9.2-22%) compared with 11% (7.8-15%) among children receiving the combination (P = 0.08, by Wilcoxon test). Seventy-three percent of chloroquine-treated children exhibited parasitemia during follow-up compared with 49% of children receiving the combination (relative risk = 1.5, 95% CI = 1.3-1.7; chi2 = 21.18, P < 0.001). A significant reduction in clinical and parasitologic treatment failure in the combination group occurred in the first two weeks following treatment, but this was eroded over weeks three and four of follow-up. The impact of combination therapy on the transmission of chloroquine-resistant parasites is discussed. Chloroquine plus artesunate is not sufficiently efficacious to justify its introduction as a replacement for chloroquine monotherapy in The Gambia

Gambian children successfully treated with chloroquine can harbor and transmit Plasmodium falciparum gametocytes carrying resistance genes.

Polymorphisms in two genes of Plasmodium falciparum (P. falciparum multidrug resistance 1 [pfmdr1] and P. falciparum chloroquine [CQ] resistance transporter [pfcrt]) are associated with CQ treatment failure. We found significant linkage disequilibrium between these loci among isolates from symptomatic Gambian children (P = 0.026) and strong selection for the resistance-associated alleles pfmdr1-86Tyr and pfcrt-76Thr in children with persistent or re-emerging P. falciparum trophozoites during post-treatment follow-up (P = 1.9 x 10(-7)). Therefore, this genotype is characteristic of resistant infections among our study population. Since the long-term public health impact of parasites carrying such resistant genotypes depends upon their transmissibility, we examined the prevalence of pfmdr1-86Tyr and pfcrt-76Thr among Gambian children harboring sexual stage parasites during post-treatment follow-up. Gametocytes that emerged after successful treatment with CQ were significantly more likely to be of this genotype than were those emerging after other treatments (P = 4.83 x 10(-4)), and were infective to Anopheles mosquitoes. Therapeutic success may thus be accompanied by public health failure as cured children pass resistance genes on to mosquitoes at an enhanced rate

Gametocytaemia after Drug Treatment of Asymptomatic Plasmodium falciparum

OBJECTIVES: Treatment of Plasmodium falciparum malaria with sulfadoxine-pyrimethamine (SP) is followed by a sharp rise in the prevalence and density of gametocytes. We did a randomized trial to determine the effect of treatment of asymptomatic infections with SP or SP plus one dose of artesunate (SP+AS) on gametocyte carriage. DESIGN: The study was a three-arm open-label randomized trial. We randomized asymptomatic carriers of P. falciparum to receive antimalarial treatment or placebo, and recorded the prevalence and density of gametocytes over the next 2 mo. SETTING: The trial was conducted during the dry (low malaria transmission) season in four rural villages in Gambia. PARTICIPANTS: Participants were adults and children aged over 6 mo with asexual P. falciparum infection and confirmed free of clinical symptoms of malaria over a 2-d screening period. INTERVENTIONS: Participants were randomized to receive a single dose of SP or SP+AS or placebo. OUTCOME MEASURES: The outcome measures were the presence of gametocytes 7 and 56 d after treatment, and the duration and density of gametocytaemia over 2 mo. RESULTS: In total, 372 asymptomatic carriers were randomized. Gametocyte prevalence on day 7 was 10.5% in the placebo group, 11.2% in the SP group (risk difference to placebo 0.7%, 95% confidence interval −7.4% to 8.7%, p = 0.87), and 7.1% in the SP+AS group (risk difference to placebo 4.1%, 95% confidence interval −3.3% to 12%, p = 0.28). By day 56, gametocyte prevalence was 13% in the placebo group and 2% in both drug-treated groups. Gametocyte carriage (the area under the curve of gametocyte density versus time), was reduced by 71% in the SP group, and by 74% in the SP+AS group, compared to placebo. Gametocyte carriage varied with age and was greater among children under 15 than among adults. CONCLUSIONS: Treatment of asymptomatic carriers of P. falciparum with SP does not increase gametocyte carriage or density. Effective treatment of asexual parasitaemia in the dry season reduces gametocyte carriage to very low levels after 4 wk

Chloroquine/Sulphadoxine-Pyrimethamine for Gambian Children with Malaria: Transmission to Mosquitoes of Multidrug-Resistant Plasmodium falciparum

OBJECTIVES: In the Gambia, chloroquine (CQ) plus sulphadoxine-pyrimethamine (SP) is the first-line antimalarial treatment. Plasmodium falciparum parasites carrying mutations associated with resistance to each of these drugs were present in 2001 but did not cause a significant loss of therapeutic efficacy among children receiving the combination CQ/SP. We measured their effect on parasite transmission to Anopheles gambiae mosquitoes. DESIGN: We conducted a single-blind, randomised, controlled trial with follow-up over 28 d. Mosquito feeding experiments were carried out 7, 10, or 14 d after treatment. SETTING: The study took place in the town of Farafenni and surrounding villages in the Gambia. PARTICIPANTS: Participants were 500 children aged 6 mo to 10 y with uncomplicated P. falciparum malaria. INTERVENTIONS: Children were randomised to receive CQ, SP, or CQ/SP. OUTCOME MEASURES: Outcomes related to transmission were determined, including posttreatment gametocyte prevalence and density. Infectiousness was assessed by membrane-feeding A. gambiae mosquitoes with blood from 70 gametocyte-positive patients. Mutations at seven loci in four genes associated with drug resistance were measured pre- and posttreatment and in the midguts of infected mosquitoes. RESULTS: After SP treatment, the infectiousness of gametocytes was delayed, compared to the other two treatment groups, despite comparable gametocyte densities. Among bloodmeal gametocytes and the midguts of infected mosquitoes, the presence of the four-locus multidrug-resistant haplotype TYRG (consisting of mutations pfcrt-76T, pfmdr1-86Y, pfdhfr-59R, and pfdhps-437G) was associated with significantly higher oocyst burdens after treatment with the combination CQ/SP. CONCLUSIONS: Parasites with a multidrug-resistant genotype had a substantial transmission advantage after CQ/SP treatment but did not have a significant impact on in vivo efficacy of this drug combination. Protocols that include measuring transmission endpoints as well as therapeutic outcomes may be a useful strategy when monitoring the evolution of drug resistance in malaria parasites in vivo

Randomised Trial of Chloroquine/Sulphadoxine-Pyrimethamine in Gambian Children with Malaria: Impact against Multidrug-Resistant P. falciparum

OBJECTIVES: In the Gambia, the combination of chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) has replaced CQ monotherapy for treatment of malaria caused by Plasmodium falciparum. We measured the efficacy of the combination CQ/SP, and the prevalence of parasites carrying alleles associated with resistance to CQ or SP. DESIGN: We conducted a single-blind, randomised, controlled trial to compare the efficacy of CQ/SP to that of SP or CQ alone. SETTING: The study took place in the town of Farafenni and surrounding villages in the Gambia. PARTICIPANTS: Participants were children aged 12 mo to 10 y presenting as outpatients with uncomplicated P. falciparum malaria. INTERVENTIONS: 500 children were randomised to receive CQ, SP, or CQ/SP as supervised treatment and actively followed over 28 d. OUTCOME MEASURES: Primary outcome was parasitaemia at any time during follow-up. Secondary outcomes were PCR-confirmed recrudescent infections among treatment failures, and clinical failure requiring rescue medication by day 28. Pretreatment parasite isolates from 161 patients were tested for the presence of resistance-associated genetic markers. RESULTS: The prevalence of parasitological failure by day 28 for the CQ group was 60.3%, compared to 17.6% for SP (odds ratio [OR], 0.106; 95% confidence interval [CI], 0.057–0.194; p < 0.001) and 13.9% for CQ/SP (OR versus CQ, 0.140; 95% CI, 0.078–0.250; p < 0.001). There was no difference between the SP and CQ/SP groups (OR, 1.324; 95% CI, 0.705–2.50). The projected prevalence of PCR-corrected treatment failure was 30.2, 6.06, and 3.94% in the CQ, SP, and CQ/SP groups, respectively. The pfdhfr-triple mutant and pfdhps-437G mutation were common, with prevalences of 67.4 and 51.2%, respectively. Pretreatment carriage of pfdhps-437G and of multidrug-resistant parasite genotypes was associated with treatment failure in the SP group, but not in the CQ or CQ/SP groups. CONCLUSIONS: The combination of CQ/SP was an efficacious treatment for uncomplicated malaria in Gambian children in this study, but the frequent occurrence of multidrug-resistant parasites suggests that this observed efficacy is not sustainable

Reduction of Malaria Transmission to Anopheles Mosquitoes with a Six-Dose Regimen of Co-Artemether

BACKGROUND: Resistance of malaria parasites to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) is increasing in prevalence in Africa. Combination therapy can both improve treatment and provide important public health benefits if it curbs the spread of parasites harbouring resistance genes. Thus, drug combinations must be identified which minimise gametocyte emergence in treated cases, and so prevent selective transmission of parasites resistant to any of the partner drugs. METHODS AND FINDINGS: In a randomised controlled trial, 497 children with uncomplicated falciparum malaria were treated with CQ and SP (three doses and one dose respectively; n = 91), or six doses of artemether in fixed combination with lumefantrine (co-artemether [Coartem, Riamet]) (n = 406). Carriage rates of Plasmodium falciparum gametocytes and trophozoites were measured 7, 14, and 28 d after treatment. The infectiousness of venous blood from 29 children carrying P. falciparum gametocytes 7 d after treatment was tested by membrane-feeding of Anopheles mosquitoes. Children treated with co-artemether were significantly less likely to carry gametocytes within the 4 weeks following treatment than those receiving CQ/SP (30 of 378 [7.94%] versus 42 of 86 [48.8%]; p < 0.0001). Carriers in the co-artemether group harboured gametocytes at significantly lower densities, for shorter periods (0.3 d versus 4.2 d; p < 0.0001) and were less infectious to mosquitoes at day 7 (p < 0.001) than carriers who had received CQ/SP. CONCLUSIONS: Co-artemether is highly effective at preventing post-treatment transmission of P. falciparum. Our results suggest that co-artemether has specific activity against immature sequestered gametocytes, and has the capacity to minimise transmission of drug-resistant parasites

The Dynamics of Naturally Acquired Immune Responses to Plasmodium falciparum Sexual Stage Antigens Pfs230 & Pfs48/45 in a Low Endemic Area in Tanzania

BACKGROUND: Naturally acquired immune responses against sexual stages of P. falciparum can reduce the transmission of malaria from humans to mosquitoes. These antigens are candidate transmission-blocking vaccines but little is known about the acquisition of sexual stage immunity after exposure to gametocytes, or their longevity and functionality. We conducted a longitudinal study on functional sexual stage immune responses. METHODOLOGY/PRINCIPAL FINDINGS: Parasitaemic individuals (n = 116) were recruited at a health centre in Lower Moshi, Tanzania. Patients presented with gametocytes (n = 16), developed circulating gametocytes by day 7 (n = 69) or between day 7 and 14 (n = 10) after treatment or did not develop gametocytes (n = 21). Serum samples were collected on the first day of gametocytaemia and 28 and 84 days post-enrolment (or d7, 28, 84 after enrolment from gametocyte-negative individuals). Antibody responses to sexual stage antigens Pfs230 and Pfs48/45 were detected in 20.7% (72/348) and 15.2% (53/348) of the samples, respectively, and were less prevalent than antibodies against asexual stage antigens MSP-1(19) (48.1%; 137/285) and AMA-1 (52.4%; 129/246)(p<0.001). The prevalence of anti-Pfs230 (p = 0.026) and anti-Pfs48/45 antibodies (p = 0.017) increased with longer duration of gametocyte exposure and had an estimated half-life of approximately 3 months. Membrane feeding experiments demonstrated a strong association between the prevalence and concentration of Pfs230 and Pfs48/45 antibodies and transmission reducing activity (TRA, p<0.01). CONCLUSIONS/SIGNIFICANCE: In a longitudinal study, anti-Pfs230 and Pfs48/45 antibodies developed rapidly after exposure to gametocytes and were strongly associated with transmission-reducing activity. Our data indicate that the extent of antigen exposure is important in eliciting functional transmission-reducing immune responses