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BackgroundGene-based vaccination using prime/boost regimens protects animals and humans against malaria, inducing cell-mediated responses that in animal models target liver stage malaria parasites. We tested a DNA prime/adenovirus boost malaria vaccine in a Phase 1 clinical trial with controlled human malaria infection.Methodology/Principal FindingsThe vaccine regimen was three monthly doses of two DNA plasmids (DNA) followed four months later by a single boost with two non-replicating human serotype 5 adenovirus vectors (Ad). The constructs encoded genes expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1). The regimen was safe and well-tolerated, with mostly mild adverse events that occurred at the site of injection. Only one AE (diarrhea), possibly related to immunization, was severe (Grade 3), preventing daily activities. Four weeks after the Ad boost, 15 study subjects were challenged with P. falciparum sporozoites by mosquito bite, and four (27%) were sterilely protected. Antibody responses by ELISA rose after Ad boost but were low (CSP geometric mean titer 210, range 44–817; AMA1 geometric mean micrograms/milliliter 11.9, range 1.5–102) and were not associated with protection. Ex vivo IFN-γ ELISpot responses after Ad boost were modest (CSP geometric mean spot forming cells/million peripheral blood mononuclear cells 86, range 13–408; AMA1 348, range 88–1270) and were highest in three protected subjects. ELISpot responses to AMA1 were significantly associated with protection (p = 0.019). Flow cytometry identified predominant IFN-γ mono-secreting CD8+ T cell responses in three protected subjects. No subjects with high pre-existing anti-Ad5 neutralizing antibodies were protected but the association was not statistically significant.SignificanceThe DNA/Ad regimen provided the highest sterile immunity achieved against malaria following immunization with a gene-based subunit vaccine (27%). Protection was associated with cell-mediated immunity to AMA1, with CSP probably contributing. Substituting a low seroprevalence vector for Ad5 and supplementing CSP/AMA1 with additional antigens may improve protection.Trial RegistrationClinicalTrials.govNCT00870987.

Mass spectrometry has revolutionized the ability to study posttranslationally modified proteoforms from biologic samples, yet we still lack methods to systematically predict, or even prioritize, which modification sites may perturb protein function. Here we describe a proteomic method to detect the effects of site-specific protein phosphorylation on the thermal stability of thousands of native proteins in live cells. This massively parallel biophysical assay unveiled shifts in overall protein stability in response to site-specific phosphorylation sites, as well as trends related to protein function and structure. This method can detect both intrinsic changes to protein structure as well as extrinsic changes to protein-protein, and protein-metabolite interactions resulting from the diminutive introduction of a phosphate onto large proteins. Finally, we show that functional "hotspot" protein modification sites can be discovered and prioritized for study in a highthroughput and unbiased fashion. This approach is applicable to diverse organisms, cell types and posttranslational modifications.

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we used single-cell sequencing to profile SARS-CoV-2-reactive B cells in 38 COVID-19 patients. Using oligo-tagged antigen baits, we isolated B cells specific to the SARS-CoV-2 spike, nucleoprotein (NP), open reading frame 8 (ORF8), and endemic human coronavirus (HCoV) spike proteins. SARS-CoV-2 spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients several months post symptom onset. With severe acute infection, substantial populations of endemic HCoV-reactive antibody-secreting cells were identified and possessed highly mutated variable genes, signifying preexisting immunity. Finally, MBCs exhibited pronounced maturation to NP and ORF8 over time, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal antibody adaptation to non-neutralizing intracellular antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.

Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique for studying the conformation dynamics and interactions of individual biomolecules. In this review, we describe the concept and principle of smFRET, illustrate general instrumentation and microscopy settings for experiments, and discuss the methods and algorithms for data analysis. Subsequently, we review applications of smFRET in protein conformational changes, ion channel open-close properties, receptor-ligand interactions, nucleic acid structure regulation, vesicle fusion, and force induced conformational dynamics. Finally, we discuss the main limitations of smFRET in molecular biology.

BackgroundFifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection.Methodology/Principal FindingsWe sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans.Conclusions/SignificanceWe suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches that elicit responses to these epitopes will increase the potency of next generation gene-based vaccines.

Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly interacts with the viral spike (S) protein of the severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2). It is proposed that inhibiting this interaction can be promising in treating COVID‐19. Here, the presence of ACE2 in extracellular vesicles (EVs) is reported and the EV‐ACE2 levels are determined by protein palmitoylation. The Cys141 and Cys498 residues on ACE2 are S‐palmitoylated by zinc finger DHHC‐Type Palmitoyltransferase 3 (ZDHHC3) and de‐palmitoylated by acyl protein thioesterase 1 (LYPLA1), which is critical for the membrane‐targeting of ACE2 and their EV secretion. Importantly, by fusing the S‐palmitoylation‐dependent plasma membrane (PM) targeting sequence with ACE2, EVs enriched with ACE2 on their surface (referred to as PM‐ACE2‐EVs) are engineered. It is shown that PM‐ACE2‐EVs can bind to the SARS‐CoV‐2 S‐RBD with high affinity and block its interaction with cell surface ACE2 in vitro. PM‐ACE2‐EVs show neutralization potency against pseudotyped and authentic SARS‐CoV‐2 in human ACE2 (hACE2) transgenic mice, efficiently block viral load of authentic SARS‐CoV‐2, and thus protect host against SARS‐CoV‐2‐induced lung inflammation. The study provides an efficient engineering protocol for constructing a promising, novel biomaterial for application in prophylactic and therapeutic treatments against COVID‐19.

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