Comprehensive, high-resolution arrays to trace the development of antibody responses to variant plasmodium falciparum antigens in naturally exposed cohorts

The National Institute of Allergy and Infectious Diseases (NIAID) awarded $451,000 to the University of California, San Francisco under the Allergy and Infectious Diseases Research program (CFDA 93.855) on August 15, 2025, with completion scheduled for July 31, 2027. This Project Grant supports the development and application of high-resolution phage immunoprecipitation sequencing (PhIP-Seq) arrays designed to comprehensively evaluate antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens and their extensive antigenic variation. The research leverages advanced bioinformatic methods and laboratory techniques to analyze naturally acquired immunity to malaria parasites by examining antibody development across the full breadth of PfEMP1 diversity.

The primary deliverable involves applying the novel PhIP-Seq array technology to well-characterized longitudinal study samples from endemic malaria regions in Uganda to characterize how antibody responses to PfEMP1 develop over time and exposure. This research addresses a significant gap in understanding naturally acquired malaria immunity by enabling high-resolution evaluation of antibody responses to the extraordinary extent of naturally occurring antigenic variation in malaria parasites, which has been previously limited by technical constraints. The resulting scientific knowledge is expected to establish groundwork for future investigations into associations between antibody responses and protective immunity against malaria disease.

Abstract

The global burden of malaria is unacceptably high and would be substantially worse without the development of naturally acquired immunity, which provides nearly complete protection against disease. Unfortunately, immunity only develops after years of exposure, in part due to extensive variation in antigens such as p. Falciparum erythrocyte membrane protein 1 (PFEMP1). PFEMP1 antigens allow the parasite to establish chronic infections and lead to host pathogenesis. Each parasite contains ~60 variable (VAR) genes encoding unique PFEMP1 sequences, which are switched during chronic infections. Antibodies to PFEMP1 are important mediators of immunity, but evaluation of antibody responses to PFEMP1 has been severely limited by an inability to capture responses to the extraordinary extent of naturally occurring antigenic variation. Furthermore, most studies have coarsely evaluated responses to large protein domains and are limited by small sample sizes, precluding the evaluation of longitudinal responses. We are uniquely positioned to address these limitations, building on our recent advances in bioinformatic and laboratory methods. We recently developed a method to produce accurate assemblies of VAR genes from short read sequence data, and a phage immunoprecipitation sequencing (phip-SEQ) assay that efficiently evaluates antibody responses to the entire p. Falciparum proteome. In this proposal, we will leverage these efforts to develop a high-resolution phip-SEQ array designed to evaluate antibody responses to the full breadth of PFEMP1 diversity. We will apply this assay to well-characterized samples from longitudinal studies of malaria in highly endemic areas of uganda to evaluate the development of antibody responses to PFEMP1, laying the groundwork for future investigations including associations of antibodies with VAR gene expression and clinical immunity. In aim 1, we will develop a phip-SEQ array for comprehensive, high-resolution profiling of antibody responses to functional domains of PFEMP1. We will create a tailored array for PFEMP1 which deeply samples global sequence diversity and evaluates epitopes at high resolution. To best capture diversity, we will augment existing VAR gene assemblies with DE novo assembly of sequences from all publicly available p. Falciparum whole genome sequences and additional sequences we are generating. We will evaluate array design approaches using well- characterized monoclonal antibodies and polyclonal immune sera. In aim 2, we will evaluate the development of antibody responses to PFEMP1 in longitudinal cohorts of children and adults naturally exposed to p. Falciparum. We will use phip-SEQ to comprehensively profile antibody responses to functional domains of PFEMP1 from infancy to adulthood by selecting cross-sectional and longitudinal time points from existing ugandan cohort samples. Upon successful completion of this study, we will have developed a low cost, high throughput method for comprehensively evaluating antibody responses to PFEMP1 and begun to answer important questions about their acquisition.