OraLee H. Branch

Genomic epidemiology of artemisinin resistant malaria.

The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 mutations have originated locally, and that kelch13 shows a normal variation pattern relative to other genes in Africa, whereas in Southeast Asia there is a great excess of non-synonymous mutations, many of which cause radical amino-acid changes. Thus, kelch13 is not currently undergoing strong selection in Africa, despite a deep reservoir of variations that could potentially allow resistance to emerge rapidly. The practical implications are that public health surveillance for artemisinin resistance should not rely on kelch13 data alone, and interventions to prevent resistance must account for local evolutionary conditions, shown by genomic epidemiology to differ greatly between geographical regions.

Plasmodium falciparum Genotypes, Low Complexity of Infection, and Resistance to Subsequent Malaria in Participants in the Asembo Bay Cohort Project†

ABSTRACT To assess the relationship between the within-host diversity of malaria infections and the susceptibility of the host to subsequent infection, we genotyped 60 childrens 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 (COIKM) (P < 0.003). Controlling for previous parasitemia, transmission, drug treatment, parasite density, sickle cell, and age, COIKM was negatively correlated with resistance to parasitemia of >500/μl (P < 0.0001). Parasitemias with the RO-genotype were more resistant than those without this genotype (P < 0.0000). The resistance in low COIKM 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 COIKM could facilitate the development of protective immunity.

A positive correlation between atypical memory B cells and Plasmodium falciparum transmission intensity in cross-sectional studies in Peru and Mali.

Background Antibodies that protect against Plasmodium falciparum (Pf) malaria are only acquired after years of repeated infections. The B cell biology that underlies this observation is poorly understood. We previously reported that “atypical” memory B cells are increased in children and adults exposed to intense Pf transmission in Mali, similar to what has been observed in individuals infected with HIV. In this study we examined B cell subsets of Pf -infected adults in Peru and Mali to determine if Pf transmission intensity correlates with atypical memory B cell expansion. Methodology/Principal Findings In this cross-sectional study venous blood was collected from adults in areas of zero (U.S., n = 10), low (Peru, n = 18) and high (Mali, n = 12) Pf transmission. Adults in Peru and Mali were infected with Pf at the time of blood collection. Thawed lymphocytes were analyzed by flow cytometry to quantify B cell subsets, including atypical memory B cells, defined by the cell surface markers CD19+ CD20+ CD21− CD27− CD10−. In Peru, the mean level of atypical memory B cells, as a percent of total B cells, was higher than U.S. adults (Peru mean: 5.4% [95% CI: 3.61–7.28]; U.S. mean: 1.4% [95% CI: 0.92–1.81]; p<0.0001) but lower than Malian adults (Mali mean 13.1% [95% CI: 10.68–15.57]; p = 0.0001). In Peru, individuals self-reporting ≥1 prior malaria episodes had a higher percentage of atypical memory B cells compared to those reporting no prior episodes (≥1 prior episodes mean: 6.6% [95% CI: 4.09–9.11]; no prior episodes mean: 3.1% [95% CI: 1.52–4.73]; p = 0.028). Conclusions/Significance Compared to Pf-naive controls, atypical memory B cells were increased in Peruvian adults exposed to low Pf transmission, and further increased in Malian adults exposed to intense Pf transmission. Understanding the origin, function and antigen specificity of atypical memory B cells in the context of Pf infection could contribute to our understanding of naturally-acquired malaria immunity.

Plasmodium falciparum and Plasmodium vivax infections in the Peruvian Amazon: propagation of complex, multiple allele-type infections without super-infection.

Outcrossing potential between Plasmodium parasites is defined by the population-level diversity (PLD) and complexity of infection (COI). There have been few studies of PLD and COI in low transmission regions. Since the 1995-1998 Peruvian Amazon epidemic, there has been sustained transmission with < 0.5 P. falciparum and < 1.6 P. vivax infections/person/year. Using weekly active case detection, we described PLD by heterozygosity (H(e)) and COI using P. falciparum Pfmsp1-B2 and P. vivax Pvmsp3alpha. Not being homologous genes, we limited comparisons to within species. P. falciparum (N = 293) had low (H(e) = 0.581) and P. vivax (N = 186) had high (H(e) = 0.845) PLD. A total of 9.5% P. falciparum infections and 26.3% P. vivax infections had COI > 1. Certain allele types were in more mixed infections than expected by chance. The few appearances of new alleles could be explained by stochastic polymerase chain reaction detection or synchronization/sequestration. The results suggest propagation of mixed infections by multiple inocula, not super-infection, implying decade-long opportunity for outcrossing in these mixed infections.

Plasmodium falciparum Genetic Diversity Maintained and Amplified Over 5 Years of a Low Transmission Endemic in the Peruvian Amazon

Plasmodium falciparum entered into the Peruvian Amazon in 1994, sparking an epidemic between 1995 and 1998. Since 2000, there has been sustained low P. falciparum transmission. The Malaria Immunology and Genetics in the Amazon project has longitudinally followed members of the community of Zungarococha (N = 1,945, 4 villages) with active household and health center-based visits each year since 2003. We examined parasite population structure and traced the parasite genetic diversity temporally and spatially. We genotyped infections over 5 years (2003–2007) using 14 microsatellite (MS) markers scattered across ten different chromosomes. Despite low transmission, there was considerable genetic diversity, which we compared with other geographic regions. We detected 182 different haplotypes from 302 parasites in 217 infections. Structure v2.2 identified five clusters (subpopulations) of phylogenetically related clones. To consider genetic diversity on a more detailed level, we defined haplotype families (hapfams) by grouping haplotypes with three or less loci differences. We identified 34 different hapfams identified. The Fst statistic and heterozygosity analysis showed the five clusters were maintained in each village throughout this time. A minimum spanning network (MSN), stratified by the year of detection, showed that haplotypes within hapfams had allele differences and haplotypes within a cluster definition were more separated in the later years (2006–2007). We modeled hapfam detection and loss, accounting for sample size and stochastic fluctuations in frequencies overtime. Principle component analysis of genetic variation revealed patterns of genetic structure with time rather than village. The population structure, genetic diversity, appearance/disappearance of the different haplotypes from 2003 to 2007 provides a genome-wide “real-time” perspective of P. falciparum parasites in a low transmission region.

Plasmodium falciparum malaria in the Peruvian Amazon, a region of low transmission, is associated with immunologic memory.

ABSTRACT The development of clinical immunity to Plasmodium falciparum malaria is thought to require years of parasite exposure, a delay often attributed to difficulties in developing protective antibody levels. In this study, we evaluated several P. falciparum vaccine candidate antigens, including apical membrane antigen 1 (AMA-1), circumsporozoite protein (CSP), erythrocyte binding antigen 175 (EBA-175), and the 19-kDa region of merozoite surface protein 1 (MSP119). After observing a more robust antibody response to MSP119, we evaluated the magnitude and longevity of IgG responses specific to this antigen in Peruvian adults and children before, during, and after P. falciparum infection. In this low-transmission region, even one reported prior infection was sufficient to produce a positive anti-MSP119 IgG response for >5 months in the absence of reinfection. We also observed an expansion of the total plasmablast (CD19+ CD27+ CD38high) population in the majority of individuals shortly after infection and detected MSP1-specific memory B cells in a subset of individuals at various postinfection time points. This evidence supports our hypothesis that effective antimalaria humoral immunity can develop in low-transmission regions.

Antibody response dynamics to the Plasmodium falciparum conserved vaccine candidate antigen, merozoite surface protein-1 C-terminal 19kD (MSP1-19kD), in Peruvians exposed to hypoendemic malaria transmission.

BackgroundIn high-transmission areas, developing immunity to symptomatic Plasmodium falciparum infections requires 2–10 years of uninterrupted exposure. Delayed malaria-immunity has been attributed to difficult-to-develop and then short-lived antibody responses.MethodsIn a study area with <0.5 P. falciparum infections/person/year, antibody responses to the MSP1-19kD antigen were evaluated and associations with P. falciparum infections in children and adults. In months surrounding and during the malaria seasons of 2003–2004, 1,772 participants received ≥6 active visits in one study-year. Community-wide surveys were conducted at the beginning and end of each malaria season, and weekly active visits were completed for randomly-selected individuals each month. There were 79 P. falciparum infections with serum samples collected during and approximately one month before and after infection. Anti-MSP1-19kD IgG levels were measured by ELISA.ResultsThe infection prevalence during February-July was similar in children (0.02–0.12 infections/person/month) and adults (0.03–0.14 infections/person/month) and was negligible in the four-month dry season. In children and adults, the seroprevalence was maintained in the beginning (children = 28.9%, adults = 61.8%) versus ending malaria-season community survey (children = 26.7%, adults = 64.6%). Despite the four-month non-transmission season, the IgG levels in Plasmodium-negative adults were similar to P. falciparum-positive adults. Although children frequently responded upon infection, the transition from a negative/low level before infection to a high level during/after infection was slower in children. Adults and children IgG-positive before infection had reduced symptoms and parasite density.ConclusionIndividuals in low transmission areas can rapidly develop and maintain αMSP1-19kD IgG responses for >4 months, unlike responses reported in high transmission study areas. A greater immune capacity might contribute to the frequent asymptomatic P. falciparum infections in this Peruvian population.

Walls talk: Microbial biogeography of homes spanning urbanization.

Home microbes track space-use and reflect a decreasing exposure to environmental microbes due to urbanization. Westernization has propelled changes in urbanization and architecture, altering our exposure to the outdoor environment from that experienced during most of human evolution. These changes might affect the developmental exposure of infants to bacteria, immune development, and human microbiome diversity. Contemporary urban humans spend most of their time indoors, and little is known about the microbes associated with different designs of the built environment and their interaction with the human immune system. This study addresses the associations between architectural design and the microbial biogeography of households across a gradient of urbanization in South America. Urbanization was associated with households’ increased isolation from outdoor environments, with additional indoor space isolation by walls. Microbes from house walls and floors segregate by location, and urban indoor walls contain human bacterial markers of space use. Urbanized spaces uniquely increase the content of human-associated microbes—which could increase transmission of potential pathogens—and decrease exposure to the environmental microbes with which humans have coevolved.

Population structure shapes copy number variation in malaria parasites

If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500 bp to 59 kb, as well as 10,107 flanking, biallelic single-nucleotide polymorphisms. Overall, CNVs were rare, small, and skewed toward low frequency variants, consistent with the deleterious model. Relative to African and South East Asian populations, CNVs were significantly more common in South America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g., DNA helicase and three conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen.

Malaria Inhibits Surface Expression of Complement Receptor 1 in Monocytes/Macrophages, Causing Decreased Immune Complex Internalization

Complement receptor 1 (CR1) expressed on the surface of phagocytic cells binds complement-bound immune complexes (IC), playing an important role in the clearance of circulating IC. This receptor is critical to prevent accumulation of IC, which can contribute to inflammatory pathology. Accumulation of circulating IC is frequently observed during malaria, although the factors contributing to this accumulation are not clearly understood. We have observed that the surface expression of CR1 on monocytes/macrophages and B cells is strongly reduced in mice infected with Plasmodium yoelii, a rodent malaria model. Monocytes/macrophages from these infected mice present a specific inhibition of complement-mediated internalization of IC caused by the decreased CR1 expression. Accordingly, mice show accumulation of circulating IC and deposition of IC in the kidneys that inversely correlate with the decrease in CR1 surface expression. Our results indicate that malaria induces a significant decrease on surface CR1 expression in the monocyte/macrophage population that results in deficient internalization of IC by monocytes/macrophages. To determine whether this phenomenon is found in human malaria patients, we have analyzed 92 patients infected with either P. falciparum (22 patients) or P. vivax (70 patients) , the most prevalent human malaria parasites. The levels of surface CR1 on peripheral monocytes/macrophages and B cells of these patients show a significant decrease compared with uninfected control individuals in the same area. We propose that this decrease in CR1 plays an essential role in impaired IC clearance during malaria.