Blood-stage malaria vaccines are intended to prevent clinical disease. The malaria vaccine FMP2.1/AS02_A, a recombinant protein based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, has previously been shown to have immunogenicity and acceptable safety in Malian adults and children. In a double-blind, randomized trial, we immunized 400 Malian children with either the malaria vaccine or a control (rabies) vaccine and followed them for 6 months. The primary end point was clinical malaria, defined as fever and at least 2500 parasites per cubic millimeter of blood. A secondary end point was clinical malaria caused by parasites with the AMA1 DNA sequence found in the vaccine strain. The cumulative incidence of the primary end point was 48.4% in the malaria-vaccine group and 54.4% in the control group; efficacy against the primary end point was 17.4% (hazard ratio for the primary end point, 0.83; 95% confidence interval [CI], 0.63 to 1.09; P=0.18). Efficacy against the first and subsequent episodes of clinical malaria, as defined on the basis of various parasite-density thresholds, was approximately 20%. Efficacy against clinical malaria caused by parasites with AMA1 corresponding to that of the vaccine strain was 64.3% (hazard ratio, 0.36; 95% CI, 0.08 to 0.86; P=0.03). Local reactions and fever after vaccination were more frequent with the malaria vaccine. On the basis of the primary end point, the malaria vaccine did not provide significant protection against clinical malaria, but on the basis of secondary results, it may have strain-specific efficacy. If this finding is confirmed, AMA1 might be useful in a multicomponent malaria vaccine. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00460525.)

We identified 480 persons with positive thick smears for asexual Plasmodium falciparum parasites, of whom 454 had positive rapid diagnostic tests (RDTs) for the histidine-rich protein 2 (HRP2) product of the hrp2 gene and 26 had negative tests. Polymerase chain reaction (PCR) amplification for the histidine-rich repeat region of that gene was negative in one-half (10/22) of false-negative specimens available, consistent with spontaneous deletion. False-negative RDTs were found only in persons with asymptomatic infections, and multiplicities of infection (MOIs) were lower in persons with false-negative RDTs (both P < 0.001). These results show that parasites that fail to produce HRP2 can cause patent bloodstream infections and false-negative RDT results. The importance of these observations is likely to increase as malaria control improves, because lower MOIs are associated with false-negative RDTs and false-negative RDTs are more frequent in persons with asymptomatic infections. These findings suggest that the use of HRP2-based RDTs should be reconsidered.

Vaccines directed against the blood stages of Plasmodium falciparum malaria are intended to prevent the parasite from invading and replicating within host cells. No blood-stage malaria vaccine has shown clinical efficacy in humans. Most malaria vaccine antigens are parasite surface proteins that have evolved extensive genetic diversity, and this diversity could allow malaria parasites to escape vaccine-induced immunity. We examined the extent and within-host dynamics of genetic diversity in the blood-stage malaria vaccine antigen apical membrane antigen–1 in a longitudinal study in Mali. Two hundred and fourteen unique apical membrane antigen–1 haplotypes were identified among 506 human infections, and amino acid changes near a putative invasion machinery binding site were strongly associated with the development of clinical symptoms, suggesting that these residues may be important to consider in designing polyvalent apical membrane antigen–1 vaccines and in assessing vaccine efficacy in field trials. This extreme diversity may pose a serious obstacle to an effective polyvalent recombinant subunit apical membrane antigen–1 vaccine.

Spatial and temporal heterogeneities in the risk of malaria have led the WHO to recommend fine-scale stratification of the epidemiological situation, making it possible to set up actions and clinical or basic researches targeting high-risk zones. Before initiating such studies it is necessary to define local patterns of malaria transmission and infection (in time and in space) in order to facilitate selection of the appropriate study population and the intervention allocation. The aim of this study was to identify, spatially and temporally, high-risk zones of malaria, at the household level (resolution of 1 to 3 m). This study took place in a Malian village with hyperendemic seasonal transmission as part of Mali-Tulane Tropical Medicine Research Center (NIAID/NIH). The study design was a dynamic cohort (22 surveys, from June 1996 to June 2001) on about 1300 children (<12 years) distributed between 173 households localized by GPS. We used the computed parasitological data to analyzed levels of Plasmodium falciparum, P. malariae and P. ovale infection and P. falciparum gametocyte carriage by means of time series and Kulldorff's scan statistic for space-time cluster detection. The time series analysis determined that malaria parasitemia (primarily P. falciparum) was persistently present throughout the population with the expected seasonal variability pattern and a downward temporal trend. We identified six high-risk clusters of P. falciparum infection, some of which persisted despite an overall tendency towards a decrease in risk. The first high-risk cluster of P. falciparum infection (rate ratio = 14.161) was detected from September 1996 to October 1996, in the north of the village. This study showed that, although infection proportions tended to decrease, high-risk zones persisted in the village particularly near temporal backwaters. Analysis of this heterogeneity at the household scale by GIS methods lead to target preventive actions more accurately on the high-risk zones identified. This mapping of malaria risk makes it possible to orient control programs, treating the high-risk zones identified as a matter of priority, and to improve the planning of intervention trials or research studies on malaria.

The disappointing efficacy of blood-stage malaria vaccines may be explained in part by allele-specific immune responses that are directed against polymorphic epitopes on blood-stage antigens. FMP2.1/AS02_A, a blood-stage candidate vaccine based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, had allele-specific efficacy against clinical malaria in a phase II trial in Malian children. We assessed the cross-protective efficacy of the malaria vaccine and inferred which polymorphic amino acid positions in AMA1 were the targets of protective allele-specific immune responses. FMP2.1/AS02_A had the highest efficacy against AMA1 alleles that were identical to the 3D7 vaccine-type allele at 8 highly polymorphic amino acid positions in the cluster 1 loop (c1L) but differed from 3D7 elsewhere in the molecule. Comparison of the incidence of vaccine-type alleles before and after vaccination in the malaria vaccine and control groups and examination of the patterns of allele change at polymorphic positions in consecutive malaria episodes suggest that the highly polymorphic amino acid position 197 in c1L was the most critical determinant of allele-specific efficacy. These results indicate that a multivalent AMA1 vaccine with broad efficacy could include only a limited set of key alleles of this extremely polymorphic antigen.

Malaria vaccines based on the 19-kDa region of merozoite surface protein 1 (MSP-1₁₉) derived from the 3D7 strain of Plasmodium falciparum are being tested in clinical trials in Africa. Knowledge of the distribution and natural dynamics of vaccine antigen polymorphisms in populations in which malaria vaccines will be tested will guide vaccine design and permit distinction between natural fluctuations in genetic diversity and vaccine-induced selection. Using pyrosequencing, six single-nucleotide polymorphisms in the nucleotide sequence encoding MSP-1₁₉ were genotyped from 1,363 malaria infections experienced by 100 children who participated in a prospective cohort study in Mali from 1999 to 2001. The frequencies of 14 MSP-1₁₉ haplotypes were compared over the course of the malaria transmission season for all three years, in three age groups, and in consecutive infections within individuals. While the frequency of individual MSP-1₁₉ haplotypes fluctuated, haplotypes corresponding to FVO and FUP strains of P. falciparum (MSP-1₁₉ haplotypes QKSNGL and EKSNGL, respectively) were most prevalent during three consecutive years and in all age groups with overall prevalences of 46% (95% confidence interval [CI] 44%–49%) and 36% (95% CI 34%–39%), respectively. The 3D7 haplotype had a lower overall prevalence of 16% (95% CI 14%–18%). Multiplicity of infection based on MSP-1₁₉ was higher at the beginning of the transmission season and in the oldest individuals (aged ≥11 y). Three MSP-1₁₉ haplotypes had a reduced frequency in symptomatic infections compared to asymptomatic infections. Analyses of the dynamics of MSP-1₁₉ polymorphisms in consecutive infections implicate three polymorphisms (at positions 1691, 1700, and 1701) as being particularly important in determining allele specificity of anti-MSP-1₁₉ immunity. Parasites with MSP-1₁₉ haplotypes different from that of the leading vaccine strain were consistently the most prevalent at a vaccine trial site. If immunity elicited by an MSP-1-based vaccine is allele-specific, a vaccine based on either the FVO or FUP strain might have better initial efficacy at this site. This study, to our knowledge the largest of its kind to date, provides molecular information needed to interpret population responses to MSP-1-based vaccines and suggests that certain MSP-1₁₉ polymorphisms may be relevant to cross-protective immunity.