Genetic diversity and malaria vaccine design, testing and efficacy: preventing and overcoming 'vaccine resistant malaria'.

The 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.

Autosome-wide linkage analysis of hip structural phenotypes in the Old Order Amish.

INTRODUCTION: Fracture risk is associated with bone mineral density (BMD) and with other indices of bone strength, including hip geometry. While the heritability and associated fracture risk of BMD are well described, less is known about genetic influences of bone geometry. We derived hip structural phenotypes using the Hip Structural Analysis program (HSA) and performed autosome-wide linkage analysis of hip geometric structural phenotypes. MATERIALS AND METHODS: The Amish Family Osteoporosis Study was designed to identify genes affecting bone health. BMD was measured at the hip using dual X-ray absorptiometry (DXA) in 879 participants (mean age+/-SD=49.8+/-16.1 years, range 18-91 years) from large multigenerational families. From DXA scans, we computed structural measures of hip geometry at the femoral neck (NN) and shaft (S) by HSA, including cross-sectional area (CSA), endocortical or inner diameter (ID), outer diameter (OD) buckling ratio (BR) and section modulus (Z). Genotyping of 731 highly polymorphic microsatellite markers (average spacing of 5.4 cM) and autosome-wide multipoint linkage analysis was performed. RESULTS: The heritability of HSA-derived hip phenotypes ranged from 40 to 84%. In the group as a whole, autosome-wide linkage analysis suggested evidence of linkage for QTLs related to NN_Z on chromosome 1p36 (LOD=2.36). In subgroup analysis, ten additional suggestive regions of linkage were found on chromosomes 1, 2, 5, 6, 11, 12, 14, 15 and 17, all with LOD>2.3 except for our linkage at 17q11.2-13 for men and women age 50 and under for NN_CSA, which had a lower LOD of 2.16, but confirmed a previous linkage report. CONCLUSIONS: We found HSA-derived measures of hip structure to be highly heritable independent of BMD. No strong evidence of linkage was found for any phenotype. Confirmatory evidence of linkage was found on chromosome 17q11.2-12 for NN_CSA. Modest evidence was found for genes affecting hip structural phenotypes at ten other chromosomal locations.

Genetic diversity in the Block 2 region of the merozoite surface protein 1 (MSP-1) of Plasmodium falciparum: additional complexity and selection and convergence in fragment size polymorphism.

Fragment size in the Block 2 repetitive region of merozoite surface protein 1 (MSP1) has commonly been used as a molecular marker in studies of malaria transmission dynamics and host immunity in Plasmodium falciparum malaria. In this study, we further explore the genetic variation in MSP-1 Block 2 underlying potential problems faced while studying the immune responses elicited by this vaccine target and while using it as a molecular marker in epidemiologic investigations. We describe the distribution of a new Block 2 recombinant allele family in samples collected from western Kenya and other malarious regions of the world and provide evidence that this allele family is found worldwide and that all MR alleles most likely originated from a single recombination event. We test whether the number of tandem repeats (i.e. fragment size) can be considered neutral in an area of high transmission in western Kenya. In addition, we investigate the validity of the assumption that Block 2 alleles of the same size and allele family are identical by examining MSP1 Block 2 amino acid sequences obtained from full-length MSP-1 clones generated from infected Kenyan children and find that this assumption does not hold. We conclude that the worldwide presence of a new allele family, the effect of positive natural selection, and the lack of conserved amino acid motifs within alleles of the same size suggest a higher level of complexity that may hamper our ability to elucidate allele family specific immune responses elicited by this vaccine target and its overall use as genetic marker in other types of epidemiologic investigations.

Plasmodium falciparum genotypes, low complexity of infection, and resistance to subsequent malaria in participants in the Asembo Bay Cohort Project.

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/microl (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.

Levels of bacterial community diversity in four arid soils compared by cultivation and 16S rRNA gene cloning.

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.

Wide distribution and diversity of members of the bacterial kingdom Acidobacterium in the environment.

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 the Acidobacterium kingdom are as genetically and metabolically diverse, environmentally widespread and perhaps as ecologically important as the well-known Proteobacteria and gram-positive bacterial kingdoms.