STRIDE: a command-line HMM-based identifier and sub-classifier of Plasmodium falciparum RIFIN and STEVOR variant surface antigen families

Background RIFINs and STEVORs are variant surface antigens expressed by P. falciparum that play roles in severe malaria pathogenesis and immune evasion. These two highly diverse multigene families feature multiple paralogs, making their classification challenging using traditional bioinformatic methods. Results STRIDE (STevor and RIfin iDEntifier) is an HMM-based, command-line program that automates the identification and classification of RIFIN and STEVOR protein sequences in the malaria parasite Plasmodium falciparum. STRIDE is more sensitive in detecting RIFINs and STEVORs than available PFAM and TIGRFAM tools and reports RIFIN subtypes and the number of sequences with a FHEYDER amino acid motif, which has been associated with severe malaria pathogenesis. Conclusions STRIDE will be beneficial to malaria research groups analyzing genome sequences and transcripts of clinical field isolates, providing insight into parasite biology and virulence.

High Antibodies to VAR2CSA in Response to Malaria Infection Are Associated With Improved Birthweight in a Longitudinal Study of Pregnant Women

IntroductionPregnant women have an increased risk of P. falciparum infection, which is associated with low birth weight and preterm delivery. VAR2CSA, a variant surface antigen expressed on the parasitized erythrocyte surface, enables sequestration in the placenta. Few studies have prospectively examined relationships between antibody responses during pregnancy and subsequent adverse birth outcomes, and there are limited data outside Africa.MethodsLevels of IgG against VAR2CSA domains (DBL3; DBL5) and a VAR2CSA-expressing placental-binding P. falciparum isolate (PfCS2-IE) were measured in 301 women enrolled at their first visit to antenatal care which occurred mid-pregnancy (median = 26 weeks, lower and upper quartiles = 22, 28). Associations between antibody levels at enrolment and placental infection, birthweight and estimated gestational age at delivery were assessed by linear and logistic regression with adjustment for confounders. For all outcomes, effect modification by gravidity and peripheral blood P. falciparum infection at enrolment was assessed.ResultsAmong women who had acquired P. falciparum infection at enrolment, those with higher levels of VAR2CSA antibodies (75th percentile) had infants with higher mean birthweight (estimates varied from +35g to +149g depending on antibody response) and reduced adjusted odds of placental infection (aOR estimates varied from 0.17 to 0.80), relative to women with lower levels (25th percentile) of VAR2CSA antibodies. However, among women who had not acquired an infection at enrolment, higher VAR2CSA antibodies were associated with increased odds of placental infection (aOR estimates varied from 1.10 to 2.24).ConclusionsWhen infected by mid-pregnancy, a better immune response to VAR2CSA-expressing parasites may contribute to protecting against adverse pregnancy outcomes.

Clinical malaria incidence following an outbreak in Ecuador was predominantly associated with Plasmodium falciparum with recombinant variant antigen gene repertoires

To better understand the factors underlying the continued incidence of clinical episodes of falciparum malaria in E-2020 countries targeting elimination, we have characterised Plasmodium falciparum disease transmission dynamics after a clonal outbreak on the northwest coast of Ecuador over a period of two years. We apply a novel, high-resolution genotyping method, the "varcode" based on a single PCR to fingerprint the DBLα region of the 40-60 members of the variant surface antigen-encoding var multigene family. Var genes are highly polymorphic within and between genomes, with var repertoires rapidly evolving by outcrossing during the obligatory sexual phase of P. falciparum in the mosquito. The continued incidence of clinical malaria after the outbreak in Ecuador provided a unique opportunity to use varcodes to document parasite microevolution and explore signatures of local disease transmission on the time scale of months to two years post-outbreak. We identified nine genetic varcodes circulating locally with spatiotemporal parasite genetic relatedness networks revealing that diversification of the clonal outbreak parasites by sexual recombination was associated with increased incidence of clinical episodes of malaria. Whether this was due to chance, immune selection or sexual recombination per se is discussed. Comparative analyses to other South American parasite populations where P. falciparum transmission remains endemic elucidated the possible origins of Ecuadorian varcodes. This analysis demonstrated that the majority of clinical cases were due to local transmission and not importation. Nonetheless, some of the varcodes that were unrelated to the outbreak varcode were found to be genetically related to other South American parasites. Our findings demonstrate the utility of the varcode as a high-resolution surveillance tool to spatiotemporally track disease outbreaks using variant surface antigen genes and resolve signatures of recombination in an E-2020 setting nearing elimination.

Mapping immune variation and var gene switching in naive hosts infected with Plasmodium falciparum

Falciparum malaria is clinically heterogeneous and the relative contribution of parasite and host in shaping disease severity remains unclear. We explored the interaction between inflammation and parasite variant surface antigen (VSA) expression, asking whether this relationship underpins the variation observed in controlled human malaria infection (CHMI). We uncovered marked heterogeneity in the host response to blood challenge; some volunteers remained quiescent, others triggered interferon-stimulated inflammation and some showed transcriptional evidence of myeloid cell suppression. Significantly, only inflammatory volunteers experienced hallmark symptoms of malaria. When we tracked temporal changes in parasite VSA expression to ask whether variants associated with severe disease rapidly expand in naive hosts, we found no transcriptional evidence to support this hypothesis. These data indicate that parasite variants that dominate severe malaria do not have an intrinsic growth or survival advantage; instead, they presumably rely upon infection-induced changes in their within-host environment for selection.

Evolutionary analyses of the major variant surface antigen-encoding genes reveal population structure of Plasmodium falciparum within and between continents

Malaria remains a major public health problem in many countries. Unlike influenza and HIV, where diversity in immunodominant surface antigens is understood geographically to inform disease surveillance, relatively little is known about the global population structure of PfEMP1, the major variant surface antigen of the malaria parasite Plasmodium falciparum. The complexity of the var multigene family that encodes PfEMP1 and that diversifies by recombination, has so far precluded its use in malaria surveillance. Recent studies have demonstrated that cost-effective deep sequencing of the region of var genes encoding the PfEMP1 DBLα domain and subsequent classification of within host sequences at 96% identity to define unique DBLα types, can reveal structure and strain dynamics within countries. However, to date there has not been a comprehensive comparison of these DBLα types between countries. By leveraging a bioinformatic approach (jumping hidden Markov model) designed specifically for the analysis of recombination within var genes and applying it to a dataset of DBLα types from 10 countries, we are able to describe population structure of DBLα types at the global scale. The sensitivity of the approach allows for the comparison of the global dataset to ape samples of Plasmodium Laverania species. Our analyses show that the evolution of the parasite population emerging out of Africa underlies current patterns of DBLα type diversity. Most importantly, we can distinguish geographic population structure within Africa between Gabon and Ghana in West Africa and Uganda in East Africa. Our evolutionary findings have translational implications in the context of globalization. Firstly, DBLα type diversity can provide a simple diagnostic framework for geographic surveillance of the rapidly evolving transmission dynamics of P. falciparum. It can also inform efforts to understand the presence or absence of global, regional and local population immunity to major surface antigen variants. Additionally, we identify a number of highly conserved DBLα types that are present globally that may be of biological significance and warrant further characterization.

Antibodies to Peptides in Semiconserved Domains of RIFINs and STEVORs Correlate with Malaria Exposure

ABSTRACT The repetitive interspersed family (RIFIN) and the subtelomeric variable open reading frame (STEVOR) family represent two of three major Plasmodium falciparum variant surface antigen families involved in malaria pathogenesis and immune evasion and are potential targets in the development of natural immunity. Protein and peptide microarrays populated with RIFINs and STEVORs associated with severe malaria vulnerability in Malian children were probed with adult and pediatric sera to identify epitopes that reflect malaria exposure. Adult sera recognized and reacted with greater intensity to all STEVOR proteins than pediatric sera did. Serorecognition of and seroreactivity to peptides within the semiconserved domain of STEVORs increased with age and seasonal malaria exposure, while serorecognition and seroreactivity increased for the semiconserved and second hypervariable domains of RIFINs only with age. Serologic responses to RIFIN and STEVOR peptides within the semiconserved domains may play a role in natural immunity to severe malaria. IMPORTANCE Malaria, an infectious disease caused by the parasite Plasmodium falciparum, causes nearly 435,000 deaths annually worldwide. RIFINs and STEVORs are two variant surface antigen families that are involved in malaria pathogenesis and immune evasion. Recent work has shown that a lack of humoral immunity to these proteins is associated with severe malaria vulnerability in Malian children. This is the first study to have compared serologic responses of children and adults to RIFINs and STEVORs in settings of malaria endemicity and to examine such serologic responses before and after a clinical malaria episode. Using microarrays, we determined that the semiconserved domains in these two parasite variant surface antigen families harbor peptides whose seroreactivity reflects malaria exposure. A similar approach has the potential to illuminate the role of variant surface antigens in the development of natural immunity to clinical malaria. Potential vaccines for severe malaria should include consideration of peptides within the semiconserved domains of RIFINs and STEVORs.