Susan K. Pierce

In an era of predicted emerging pandemics, the production of effective vaccines may require an in-depth understanding of the biology of human naive B (B_N) cells. Here we provide evidence that the majority of B_N cells expressed CD73, an ecto-5’-nucleotidase often associated with immune cell suppression, and demonstrated features of anergy, including an IgM^lowIgD⁺ surface phenotype, reduced calcium flux in response to IgM crosslinking, and increased PTEN expression. Analysis of antibody sequences encoded by the inherently autoreactive V_H4-34 heavy chain produced by plasmablasts seven days following influenza vaccination showed that in younger but not in older individuals, anergic B_N cells provided a reservoir of B cells capable of responding to vaccination by somatic mutation, resulting in diversification and loss of autoreactivity. These results suggest that effective human vaccines may require the ability to awaken or ‘redeem’ anergic B_N cells that can be repurposed to participate in pathogen-specific responses.

B cell responses to membrane-presented antigens appear to be strongly favored over soluble antigens in vivo suggesting that vaccines that mimic membrane-presented antigens may be highly efficacious. We recently demonstrated that human B cell responses to membrane-associated but not to soluble antigens in vitro depended on the expression and activity of the plasma membrane mechanosensitive ion channel, Piezo1. Here, we provide evidence that the efficacy of the current human papillomavirus virus-like particle (HPV VLP) vaccines may be due in part to their inherent ability to mimic Piezo1-dependent membrane presentation of antigens to B cells. We compared HPV-specific human B cell responses to HPV VLPs versus soluble HPV pentameric capsomeres and showed that although both induced calcium responses, only HPV VLP-induced responses were blocked by Piezo1 inhibitors. The kinetics of internalization of HPV-VLP and capsomeres into HPV-specific B cells were similar and neither required Piezo1 function as shown by small interfering RNA (siRNA)-mediated knockdown of Piezo. However, trafficking of HPV-VLPs into intracellular major histocompatibility complex (MHC) class II, lysosomal associated membrane protein 1 (LAMP1) ⁺ antigen-processing compartments was Piezo1-dependent, whereas trafficking of capsomeres was not. In addition, a time course of intracellular trafficking suggested that colocalization of HPV-VLP with MHC classII was more stable over time as compared to capsomeres. Taken together these findings suggest that the ability of HPV-VLP vaccines to mimic Piezo1-dependent membrane antigen presentation may be exploited in the design of highly effective human vaccines.

Background: Despite treatment with highly effective antimalarial drugs, malaria annually claims the lives of over half a million children under 5-years of age in sub-Saharan Africa. Cerebral malaria (CM), defined as Plasmodium falciparum infection with coma, is the severe malaria syndrome with the highest mortality. Studies in the CM mouse model suggest that a T cell-mediated response underlies CM pathology, opening a new target for therapy in humans. This trial aims to establish the preliminary safety of one such novel therapy, the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON). Methods: In this phase I/IIa dose-escalation clinical trial, a single dose of intravenous (IV) DON is administered to three participants groups—healthy adults and adults with uncomplicated malaria, then pediatric participants with CM—to primarily assess safety. The secondary objective of this trial is to assess pharmacokinetics of DON over a range of doses. The open-label adult portion of the trial enrolls 40 healthy adults concurrently with 40 adults with uncomplicated malaria. Cohorts of 10 participants receive a single IV dose of DON with doses escalating between cohorts from 0.1 mg/kg, 1.0 mg/kg, 5.0 mg/kg, to 10 mg/kg. Following subsequent safety review, a randomized, double-blind, and placebo-controlled pediatric study enrolls 72 participants aged 6 months to 14 years with CM. The pediatric portion of the study minimally spans three malaria seasons including a planned interim analysis after 50% of pediatric enrollments. The first half of pediatric participants receive DON 0.1 mg/kg, 1.0 mg/kg, or placebo. Dosing for the second half of pediatric participants is informed by the safety and preliminary efficacy results of those previously enrolled. The pediatric portion of the study has an exploratory outcome evaluating the preliminary efficacy of DON. Efficacy is assessed by diagnostics predictive of CM outcome: electroencephalography (EEG), magnetic resonance imaging (MRI), and transcranial doppler (TCD), measured before and after DON administration. All participants with malaria receive standard of care antimalarials in accordance with local guidelines, regardless of study drug dose group. Discussion: This preliminary safety and efficacy study evaluates DON, a candidate adjunctive therapy for pediatric CM. If results support DON preliminary safety and efficacy, follow-up phase II and III clinical trials will be indicated. Trial registration: This trial was registered on ClinicalTrials.gov on 28 July 2022 (NCT05478720).

The demand for a vaccine for coronavirus disease 2019 (COVID-19) highlighted gaps in our understanding of the requirements for B cell responses to antigens, particularly to membrane-presented antigens, as occurs in vivo. We found that human B cell responses to membrane-presented antigens required the function of Piezo1, a plasma membrane mechanosensitive cation channel. Simply making contact with a glass probe induced calcium (Ca ²⁺ ) fluxes in B cells that were blocked by the Piezo1 inhibitor GsMTx4. When placed on glass surfaces, the plasma membrane tension of B cells increased, which stimulated Ca ²⁺ influx and spreading of B cells over the glass surface, which was blocked by the Piezo1 inhibitor OB-1. B cell responses to membrane-presented antigens but not to soluble antigens were inhibited both by Piezo1 inhibitors and by siRNA-mediated knockdown of Piezo1. Thus, the activation of Piezo1 defines an essential event in B cell activation to membrane-presented antigens that may be exploited to improve the efficacy of vaccines.

Molecular structures provide a road map for understanding and controlling B cell receptor activation

Chronic infectious diseases have a substantial impact on the human B cell compartment including a notable expansion of B cells here termed atypical B cells (ABCs). Using unbiased single-cell RNA sequencing (scRNA-seq), we uncovered and characterized heterogeneities in naïve B cell, classical memory B cells, and ABC subsets. We showed remarkably similar transcriptional profiles for ABC clusters in malaria, HIV, and autoimmune diseases and demonstrated that interferon-γ drove the expansion of ABCs in malaria. These observations suggest that ABCs represent a separate B cell lineage with a common inducer that further diversifies and acquires disease-specific characteristics and functions. In malaria, we identified ABC subsets based on isotype expression that differed in expansion in African children and in B cell receptor repertoire characteristics. Of particular interest, IgD⁺IgM^lo and IgD⁻IgG⁺ ABCs acquired a high antigen affinity threshold for activation, suggesting that ABCs may limit autoimmune responses to low-affinity self-antigens in chronic malaria.

Cerebral malaria (CM) is the deadliest form of severe Plasmodium infections. Currently, we have limited understanding of the mechanisms by which Plasmodium parasites induce CM. The mouse model of CM, experimental CM (ECM), induced by infection with the rodent parasite, Plasmodium berghei ANKA (PbANKA) has been extensively used to study the pathophysiology of CM. Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65), that does not cause ECM, differ in only 21 single nucleotide polymorphysims (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. Although the majority of these SNPs are located in genes of unknown function, one SNP is located in the DNA binding site of a member of the Plasmodium ApiAP2 transcription factor family, that we recently showed functions as a virulence factor alternating the host’s immune response to the parasite. Here, we investigated the impact of this SNP on the development of ECM. Our results using CRISPR-Cas9 engineered parasites indicate that despite its immune modulatory function, the SNP is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in ECM phenotypes.

Atypical memory B cell antigens must be membrane associated to force exclusion of inhibitory receptors from the immune synapse.