Chloroquine is again an efficacious treatment for malaria, 12 years after it was withdrawn from use in Malawi. (ClinicalTrials.gov number, NCT00125489 [ClinicalTrials.gov].).

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. †To whom correspondence should be addressed. cplowe@medicine.umaryland.edu. * Present address: University of Maryland Dental School, Baltimore, MD 21201, USA. Author contributions: S.L.T. conceived the study concept, assisted with the sequencing, analyzed the data, and wrote the paper. D.C., M.A.T., K.T., A.N., and O.K.D. designed and conducted the malaria incidence study. A.H.B. determined the crystal structure of the AMA-1 protein and contributed to writing the paper. M.P.C. and A.A.E. assisted with the data analysis and contributed to writing the paper. A.O. assisted with the sequencing and contributed to writing the paper. A.A.D. contributed to writing the paper. C.V.P. designed the malaria incidence study, helped develop the study concept, assisted with the data analysis, contributed to writing the paper, and provided funding for study activities. SUPPLEMENTARY MATERIAL

To assess the distribution and diversity of members of the recently identified bacterial kingdom Acidobacterium, members of this kingdom present in 43 environmental samples were surveyed by PCR amplification. A primer designed to amplify rRNA gene sequences (ribosomal DNAs [rDNAs]) from most known members of the kingdom was used to interrogate bulk DNA extracted from the samples. Positive PCR results were obtained with all temperate soil and sediment samples tested, as well as some hot spring samples, indicating that members of this kingdom are very widespread in terrestrial environments. PCR primers specific for four phylogenetic subgroups within the kingdom were used in similar surveys. All four subgroups were detected in most neutral soils and some sediments, while only two of the groups were seen in most low-pH environments. The combined use of these primers allowed identification of a novel lineage within the kingdom in a hot spring environment. Phylogenetic analysis of rDNA sequences from our survey and the literature outlines at least six major subgroups within the kingdom. Taken together, these data suggest that members of theAcidobacterium kingdom are as genetically and metabolically diverse, environmentally widespread and perhaps as ecologically important as the well-known Proteobacteria and gram-positive bacterial kingdoms.

SUMMARYThe development of effective malaria vaccines may be hindered by extensive genetic diversity in the surface proteins being employed as vaccine antigens. Understanding of the extent and dynamics of genetic diversity in vaccine antigens is needed to guide rational vaccine design and to interpret the results of vaccine efficacy trials conducted in malaria endemic areas. Molecular epidemiological, population genetic, and structural approaches are being employed to try to identify immunologically relevant polymorphism in vaccine antigens. The results of these studies will inform choices of which alleles to include in multivalent or chimeric vaccines; however, additional molecular and immuno-epidemiological studies in a variety of geographic locations will be necessary for these approaches to succeed. Alternative means of overcoming antigenic diversity are also being explored, including boosting responses to critical conserved regions of current vaccine antigens, identifying new, more conserved and less immunodominant antigens, and developing whole-organism vaccines. Continued creative application and integration of tools from multiple disciplines, including epidemiology, immunology, molecular biology, and evolutionary genetics and genomics, will likely be required to develop broadly protective vaccines against Plasmodium and other antigenically complex pathogens.

Background: Insecticide Treated Nets (ITNs) are an important tool for malaria control. ITNs are effective because they work on several parts of the mosquito feeding cycle, including both adult killing and repelling effects.

Techniques based on amplification of 16S rRNA genes for comparing bacterial communities are now widely used in microbial ecology, but calibration of these techniques with traditional tools, such as cultivation, has been conspicuously absent. In this study, we compared levels of bacterial community diversity in two pinyon rhizosphere soil samples and two between-tree (interspace) soil samples by analyzing 179 cultivated bacterial isolates and 801 16S rRNA genes amplified from extracted soil DNA. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes RsaI and BstUI. The average level of 16S rRNA gene sequence similarity of members of a phylotype was 86.6% based on an analysis of partial sequences. A total of 498 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, while 34 phylotypes occurred among the cultivated isolates. Analysis of sequences from a subset of the phylotypes showed that at least seven bacterial divisions were represented in the clone libraries, whereas the isolates represented only three. The phylotype richness, frequency distribution (evenness), and composition of the four culture collections and the four clone libraries were investigated by using a variety of diversity indices. Although cultivation and 16S rRNA cloning analyses gave contradictory descriptions of the relative phylotype richness for one of the four environments, the two methods identified qualitatively consistent relationships when levels of evenness were compared. The levels of phylotype similarity between communities were uniformly low (15 to 31%). Both methods consistently indicated that one environment was distinct from the other three. Our data illustrate that while 16S rDNA cloning and cultivation generally describe similar relationships between soil microbial communities, significant discrepancies can occur.

BackgroundMalaria 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.Methods and FindingsUsing 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.ConclusionsParasites 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.

To assess the relationship between the within-host diversity of malaria infections and the susceptibility of the host to subsequent infection, we genotyped 60 children's successive infections from birth through 3 years of life. MSP-1 Block2 genotypes were used to estimate the complexity of infection (COI). Malaria transmission and age were positively associated with the number of K1 and Mad20 alleles detected (COI KM ) (P < 0.003). Controlling for previous parasitemia, transmission, drug treatment, parasite density, sickle cell, and age, COI KM was negatively correlated with resistance to parasitemia of >500/l (P < 0.0001). Parasitemias with the RO-genotype were more resistant than those without this genotype (P < 0.0000). The resistance in low COI KM infections was not genotype specific. We discuss the impact of genotype-transcending immunity to conserved antigenic determinants. We also propose a diversity-driven immunomodulation hypothesis that may explain the delayed development of natural immunity in the first few years of life and suggest that interventions that decrease the COI KM could facilitate the development of protective immunity.