Linkage Group Selection: Towards Identifying Genes Controlling Strain Specific Protective Immunity in Malaria

Protective immunity against blood infections of malaria is partly specific to the genotype, or strain, of the parasites. The target antigens of Strain Specific Protective Immunity are expected, therefore, to be antigenically and genetically distinct in different lines of parasite. Here we describe the use of a genetic approach, Linkage Group Selection, to locate the target(s) of Strain Specific Protective Immunity in the rodent malaria parasite Plasmodium chabaudi chabaudi. In a previous such analysis using the progeny of a genetic cross between P. c. chabaudi lines AS-pyr1 and CB, a location on P. c. chabaudi chromosome 8 containing the gene for merozoite surface protein-1, a known candidate antigen for Strain Specific Protective Immunity, was strongly selected. P. c. chabaudi apical membrane antigen-1, another candidate for Strain Specific Protective Immunity, could not have been evaluated in this cross as AS-pyr1 and CB are identical within the cell surface domain of this protein. Here we use Linkage Group Selection analysis of Strain Specific Protective Immunity in a cross between P. c. chabaudi lines CB-pyr10 and AJ, in which merozoite surface protein-1 and apical membrane antigen-1 are both genetically distinct. In this analysis strain specific immune selection acted strongly on the region of P. c. chabaudi chromosome 8 encoding merozoite surface protein-1 and, less strongly, on the P. c. chabaudi chromosome 9 region encoding apical membrane antigen-1. The evidence from these two independent studies indicates that Strain Specific Protective Immunity in P. c. chabaudi in mice is mainly determined by a narrow region of the P. c. chabaudi genome containing the gene for the P. c. chabaudi merozoite surface protein-1 protein. Other regions, including that containing the gene for P. c. chabaudi apical membrane antigen-1, may be more weakly associated with Strain Specific Protective Immunity in these parasites

Stimulus Dependence of Barrel Cortex Directional Selectivity

Neurons throughout the rat vibrissa somatosensory pathway are sensitive to the angular direction of whisker movement. Could this sensitivity help rats discriminate stimuli? Here we use a simple computational model of cortical neurons to analyze the robustness of directional selectivity. In the model, directional preference emerges from tuning of synaptic conductance amplitude and latency, as in recent experimental findings. We find that directional selectivity during stimulation with random deflection sequences is strongly dependent on the mean deflection frequency: Selectivity is weakened at high frequencies even when each individual deflection evokes strong directional tuning. This variability of directional selectivity is due to generic properties of synaptic integration by the neuronal membrane, and is therefore likely to hold under very general physiological conditions. Our results suggest that directional selectivity depends on stimulus context. It may participate in tasks involving brief whisker contact, such as detection of object position, but is likely to be weakened in tasks involving sustained whisker exploration (e.g., texture discrimination)

Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients.

Naturally acquired blood-stage infections of the malaria parasite Plasmodium falciparum typically harbour multiple haploid clones. The apparent number of clones observed in any single infection depends on the diversity of the polymorphic markers used for the analysis, and the relative abundance of rare clones, which frequently fail to be detected among PCR products derived from numerically dominant clones. However, minority clones are of clinical interest as they may harbour genes conferring drug resistance, leading to enhanced survival after treatment and the possibility of subsequent therapeutic failure. We deployed new generation sequencing to derive genome data for five non-propagated parasite isolates taken directly from 4 different patients treated for clinical malaria in a UK hospital. Analysis of depth of coverage and length of sequence intervals between paired reads identified both previously described and novel gene deletions and amplifications. Full-length sequence data was extracted for 6 loci considered to be under selection by antimalarial drugs, and both known and previously unknown amino acid substitutions were identified. Full mitochondrial genomes were extracted from the sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data. Finally, genome-wide analysis of clone multiplicity was performed, and the number of infecting parasite clones estimated for each isolate. Each patient harboured at least 3 clones of P. falciparum by this analysis, consistent with results obtained with conventional PCR analysis of polymorphic merozoite antigen loci. We conclude that genome sequencing of peripheral blood P. falciparum taken directly from malaria patients provides high quality data useful for drug resistance studies, genomic structural analyses and population genetics, and also robustly represents clonal multiplicity

Increased sensitivity for detecting malaria parasites in human umbilical cord blood using scaled-up DNA preparation

BACKGROUND: All mothers donating umbilical cord blood units to the NHS cord blood bank undergo an assessment for the likelihood of prior exposure to malaria infection. Those deemed at risk due to a history of travel to, or residence in, malaria endemic regions are screened serologically to detect anti-malaria antibodies. A positive result excludes the use of the cord blood for transplant therapy unless a risk assessment can ensure that malaria transmission is extremely unlikely. This paper details the screening of cord blood units from malaria serology positive mothers to detect malaria parasite DNA using a highly sensitive nested PCR. METHODS: Uninfected blood from a healthy volunteer was spiked with known quantities of malaria parasites and 5 millilitre and 200 microlitre aliquots were subjected to DNA extraction using QIAamp DNA maxi and DNA mini kits respectively. Nested PCR, to detect malarial SSU rRNA sequences, was performed on the purified DNA samples to determine the limit of detection for this assay with both extraction methodologies. Following assay validation, 54 cord blood units donated by mothers who were positive for anti-malaria antibodies were screened by this approach. RESULTS: When DNA was purified from 5 millilitres of blood it was possible to routinely detect as few as 50 malaria parasites per millilitre using nested PCR. This equates to a significant increase in the sensitivity of the current gold standard nucleic acid amplification technique used to detect malaria parasites (routinely performed from > 200 microlitre volumes of blood). None of the 54 donated cord blood units from serology positive mothers tested positive for malaria parasites using this scaled up DNA preparation method. CONCLUSION: Serological testing for malaria parasites may be overly conservative, leading to unnecessary rejection of cord blood donations that lack malaria parasites and which are, therefore, safe for use in stem cell therapy

A novel malaria vaccine candidate antigen expressed in Tetrahymena thermophila

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens

Contrasting Population Structures of the Genes Encoding Ten Leading Vaccine-Candidate Antigens of the Human Malaria Parasite, Plasmodium falciparum

The extensive diversity of Plasmodium falciparum antigens is a major obstacle to a broadly effective malaria vaccine but population genetics has rarely been used to guide vaccine design. We have completed a meta-population genetic analysis of the genes encoding ten leading P. falciparum vaccine antigens, including the pre-erythrocytic antigens csp, trap, lsa1 and glurp; the merozoite antigens eba175, ama1, msp's 1, 3 and 4, and the gametocyte antigen pfs48/45. A total of 4553 antigen sequences were assembled from published data and we estimated the range and distribution of diversity worldwide using traditional population genetics, Bayesian clustering and network analysis. Although a large number of distinct haplotypes were identified for each antigen, they were organized into a limited number of discrete subgroups. While the non-merozoite antigens showed geographically variable levels of diversity and geographic restriction of specific subgroups, the merozoite antigens had high levels of diversity globally, and a worldwide distribution of each subgroup. This shows that the diversity of the non-merozoite antigens is organized by physical or other location-specific barriers to gene flow and that of merozoite antigens by features intrinsic to all populations, one important possibility being the immune response of the human host. We also show that current malaria vaccine formulations are based upon low prevalence haplotypes from a single subgroup and thus may represent only a small proportion of the global parasite population. This study demonstrates significant contrasts in the population structure of P. falciparum vaccine candidates that are consistent with the merozoite antigens being under stronger balancing selection than non-merozoite antigens and suggesting that unique approaches to vaccine design will be required. The results of this study also provide a realistic framework for the diversity of these antigens to be incorporated into the design of next-generation malaria vaccines

Sequence Conservation in Plasmodium falciparum α-Helical Coiled Coil Domains Proposed for Vaccine Development

BACKGROUND: The availability of the P. falciparum genome has led to novel ways to identify potential vaccine candidates. A new approach for antigen discovery based on the bioinformatic selection of heptad repeat motifs corresponding to alpha-helical coiled coil structures yielded promising results. To elucidate the question about the relationship between the coiled coil motifs and their sequence conservation, we have assessed the extent of polymorphism in putative alpha-helical coiled coil domains in culture strains, in natural populations and in the single nucleotide polymorphism data available at PlasmoDB. METHODOLOGY/PRINCIPAL FINDINGS: 14 alpha-helical coiled coil domains were selected based on preclinical experimental evaluation. They were tested by PCR amplification and sequencing of different P. falciparum culture strains and field isolates. We found that only 3 out of 14 alpha-helical coiled coils showed point mutations and/or length polymorphisms. Based on promising immunological results 5 of these peptides were selected for further analysis. Direct sequencing of field samples from Papua New Guinea and Tanzania showed that 3 out of these 5 peptides were completely conserved. An in silico analysis of polymorphism was performed for all 166 putative alpha-helical coiled coil domains originally identified in the P. falciparum genome. We found that 82% (137/166) of these peptides were conserved, and for one peptide only the detected SNPs decreased substantially the probability score for alpha-helical coiled coil formation. More SNPs were found in arrays of almost perfect tandem repeats. In summary, the coiled coil structure prediction was rarely modified by SNPs. The analysis revealed a number of peptides with strictly conserved alpha-helical coiled coil motifs. CONCLUSION/SIGNIFICANCE: We conclude that the selection of alpha-helical coiled coil structural motifs is a valuable approach to identify potential vaccine targets showing a high degree of conservation

Mild Sensory Stimulation Completely Protects the Adult Rodent Cortex from Ischemic Stroke

Despite progress in reducing ischemic stroke damage, complete protection remains elusive. Here we demonstrate that, after permanent occlusion of a major cortical artery (middle cerebral artery; MCA), single whisker stimulation can induce complete protection of the adult rat cortex, but only if administered within a critical time window. Animals that receive early treatment are histologically and behaviorally equivalent to healthy controls and have normal neuronal function. Protection of the cortex clearly requires reperfusion to the ischemic area despite permanent occlusion. Using blood flow imaging and other techniques we found evidence of reversed blood flow into MCA branches from an alternate arterial source via collateral vessels (inter-arterial connections), a potential mechanism for reperfusion. These findings suggest that the cortex is capable of extensive blood flow reorganization and more importantly that mild sensory stimulation can provide complete protection from impending stroke given early intervention. Such non-invasive, non-pharmacological intervention has clear translational potential

The Breadth, but Not the Magnitude, of Circulating Memory B Cell Responses to P. falciparum Increases with Age/Exposure in an Area of Low Transmission

BACKGROUND: Malaria caused by Plasmodium falciparum remains a major cause of death in sub-Saharan Africa. Immunity against symptoms of malaria requires repeated exposure, suggesting either that the parasite is poorly immunogenic or that the development of effective immune responses to malaria may be impaired. METHODS: We carried out two age-stratified cross-sectional surveys of anti-malarial humoral immune responses in a Gambian village where P. falciparum malaria transmission is low and sporadic. Circulating antibodies and memory B cells (MBC) to four malarial antigens were measured using ELISA and cultured B cell ELISpot. FINDINGS AND CONCLUSIONS: The proportion of individuals with malaria-specific MBC and antibodies, and the average number of antigens recognised by each individual, increased with age but the magnitude of these responses did not. Malaria-specific antibody levels did not correlate with either the prevalence or median number of MBC, indicating that these two assays are measuring different aspects of the humoral immune response. Among those with immunological evidence of malaria exposure (defined as a positive response to at least one malarial antigen either by ELISA or ELISPOT), the median number of malaria-specific MBC was similar to median numbers of diphtheria-specific MBC, suggesting that the circulating memory cell pool for malaria antigens is of similar size to that for other antigens