Biology subjects

King, C. R.

Publications and source records attributed to King, C. R..

17 recordsLinked to original sources

bioRxiv2026

Virus-Driven Proximity Proteomics Identifies p400 as a Restriction Factor Poised on Incoming Viral Genomes

Immediate-early viral proteins must rapidly counteract host defenses directed at incoming viral genomes, yet the protein interaction networks that mediate this remain unresolved. This gap persists largely because immediate-early proteins are expressed at low abundance during this stage of infection, making them challenging to investigate with standard approaches. Here, we present a virus-driven proximity proteomics framework to investigate immediate-early phase virus-host interactions in an authentic infection context. An isogenic P2A control virus untethers a miniTurbo biotin ligase from the viral protein under investigation, effectively modeling bait abundance and background changes from a matched infection context. Continuous biotin labeling from the earliest hours of infection further amplifies detection of transient, low-abundance interactions characteristic of this phase. Using the adenovirus E1A hub protein as a benchmark, we recovered the majority of known E1A interactors and identified over 150 high-confidence interactions. Applying this strategy to the immediate-early phase, we resolved the E1A interactome and identified the p400 chromatin remodeling complex as its dominant target. We show p400 associates with incoming viral genomes, represses gene expression, and regulates persistent infection. These data identify p400 as a poised host restriction factor and establish a temporally resolved proximity proteomics strategy transferable to other immediate-early viral proteins.

microbiology↗

bioRxiv2025

Anti-malaria RTS,S/AS01 vaccine generates a limited pool of memory B cells expressing specificities associated with protection

RTS,S/AS01, an adjuvanted subunit malaria vaccine, induces protective but short-lived anti- Plasmodium falciparum circumsporozoite protein (CSP) antibody titers. To better understand the lack of sustained protection post-vaccination, we analyzed CSP-specific B cells over time in malaria-naive individuals following immunization with RTS,S/AS01. Longitudinal analyses of the cellular responses revealed a shift in the specificity of CSP-specific B cells over time. Early post- vaccine responses were dominated by plamsablasts and class-switched memory B cells (MBCs) specific for the NANP-repeat region of CSP, epitopes associated with protective antibodies. However, the frequency of NANP-repeat-specific MBCs declines, while longer-term memory specific for the C-terminus of CSP persists. Despite being class-switched and affinity matured, C- term monoclonal antibodies derived from these MBCs failed to protect mice against a transgenic parasite challenge. Taken together, these findings suggest that RTS,S/AS01 induces a transient, protective NANP repeat-specific B cell response that is subsequently replaced by memory B cells with non-protective reactivities, potentially explaining its limited long-term efficacy and limited response to subsequent challenges or boosters.

immunology↗

bioRxiv2025

Bigger Is Better Than Many: A Strategy to Optimize Multi-Gene Co-Expression

Large plasmids are often avoided in mammalian co-transfection due to the assumption that they transfect poorly, driving the use of multiple smaller plasmids. Here, we pair finite-state-projection modeling with flow cytometry experiments to compare one-, two-, and three-plasmid delivery of GFP/BFP/RFP. Estimated entry rates were size-independent from 4.9 to 16.4 kb, indicating that plasmid length is not the dominant barrier in this range. Our results suggest that using lipofectamine slightly increases co-transfection efficiency due to the ability of lipoplexes to contain multiple plasmids. However, this benefit is limited to only delivering two plasmids. Additionally, we show that contrary to current beliefs, putting all genes onto the same plasmid both increases the probability that a cell will express all genes of interest and results in a tighter correlation of gene expression levels compared to these multi-plasmid systems. Together, these results identify multi-cargo delivery and not plasmid size as the key constraint on co-transfection and show that single-plasmid designs are generally preferable for applications such as viral-vector production. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/675629v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@251928org.highwire.dtl.DTLVardef@196d1cborg.highwire.dtl.DTLVardef@a781e9org.highwire.dtl.DTLVardef@1420928_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

IFIT3 RNA-binding activity promotes influenza A virus infection and translation efficiency

Host cells produce a vast network of antiviral factors in response to viral infection. The interferon-induced proteins with tetratricopeptide repeats (IFITs) are important effectors of a broad-spectrum antiviral response. In contrast to their canonical roles, we previously identified IFIT2 and IFIT3 as pro-viral host factors during influenza A virus (IAV) infection. During IAV infection, IFIT2 binds and enhances translation of AU-rich cellular mRNAs, including many IFN-simulated gene products, establishing a model for its broad antiviral activity. But, IFIT2 also bound viral mRNAs and enhanced their translation resulting in increased viral replication. The ability of IFIT3 to bind RNA and whether this is important for its function was not known. Here we validate direct interactions between IFIT3 and RNA using electromobility shift assays (EMSAs). RNA-binding site identification (RBS-ID) experiments then identified an RNA-binding surface composed of residues conserved in IFIT3 orthologs and IFIT2 paralogs. Mutation of the RNA-binding site reduced the ability IFIT3 to promote IAV gene expression and translation efficiency when compared to wild type IFIT3. The functional units of IFIT2 and IFIT3 are homo- and heterodimers, however the RNA-binding surfaces are located near the dimerization interface. Using co-immunoprecipitation, we showed that mutations to these sites do not affect dimerization. Together, these data establish the link between IFIT3 RNA-binding and its ability to modulate translation of host and viral mRNAs during IAV infection. ImportanceInfluenza A viruses (IAV) cause considerable morbidity and mortality through sporadic pandemics as well as annual epidemics. Zoonotic IAV strains pose an additional risk of spillover into a naive human population where prior immunity can have minimal effect. In this case, the first line of defense in the host is the innate immune response. Interferon stimulated genes (ISGs) produce a suite of proteins that are front-line effectors of innate immune responses. While ISGs are typically considered antiviral, new work has revealed an emerging trend where viruses co-opt ISGs for pro-viral function. Here, we determine how the ISG IFIT3 is used by IAV as a pro-viral factor, advancing our understanding of IFIT3 function generally as well as specifically in the context of IAV infection.

microbiology↗

bioRxiv2024

The Transcriptional Gradient in Negative-Strand RNA Viruses Suggests a Common RNA Transcription Mechanism

We introduce a novel model of nonsegmented negative-strand RNA virus (NNSV) transcription. Previous models have relied on polymerase behavioral differences in the highly conserved intergenic sequences. Our model hypothesizes the transcriptional gradient in NNSVs is explained through a simple model with two parameters associated with the viral polymerase. Most differences in expression can be attributed to the processivity of the polymerase while additional attenuation occurs in the presence of overlapping genes. This model reveals a correlation between polymerase processivity and genome length, which is consistent with the universal entry of polymerases through the 3 end of the genome. Using this model, it is now possible to predict the transcriptional behavior of NNSVs from genotype alone, revolutionizing the design of novel NNSV variants for biomedical applications.

bioinformatics↗

bioRxiv2024

Repertoire, function, and structure of serological antibodies induced by the R21/Matrix-M malaria vaccine

The World Health Organization recently recommended the programmatic use of R21/Matrix-M vaccine for Plasmodium falciparum malaria prevention in children living in malaria-endemic areas. To determine its effects on humoral immunity, we conducted a proteomic analysis of polyclonal IgG antibodies directed against the NANP tetrapeptide of the circumsporozoite protein (CSP) which comprises the vaccines core immunogen. In ten malaria-naive adult volunteers, R21/Matrix-M induced polarized IgG anti-NANP repertoires, heavily skewed for IGHV3-30/3-33 genes bearing minimal somatic mutation, which remained static in composition following a controlled human malaria infection challenge. Notably, these vaccine-generated antibodies cross-reacted with another protective CSP epitope, the N-terminal junction region, despite its absence from the R21 construct. NANP-specific IGHV3-30/3-33 monoclonal antibodies mined from polyclonal IgG repertoires blocked sporozoite invasion in vitro and prevented parasitemia in vivo. Overall, R21/Matrix-M elicits polarized, minimally mutated, polyclonal IgG responses that can target multiple protective CSP epitopes, offering molecular insight into the serological basis for its demonstrated efficacy against P. falciparum malaria.

immunology↗

bioRxiv2024

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex

In recent years, reticulocyte-binding protein homologue 5 (RH5) has emerged as a leading blood-stage Plasmodium falciparum malaria vaccine antigen. The most advanced blood-stage vaccine candidate in a Phase 2b clinical trial, RH5.1/Matrix-M, is based on a full-length soluble protein-with-adjuvant formulation. RH5 interacts with cysteine-rich protective antigen (CyRPA) and RH5- interacting protein (RIPR) to form an essential heterotrimeric "RCR-complex". Here, we investigated whether a vaccine candidate based on the ternary RCR-complex could substantially improve upon the leading clinical candidate RH5.1/Matrix-M in preclinical studies. Using a panel of monoclonal antibodies (mAbs) we confirm that parasite growth-inhibitory epitopes on each antigen are exposed on the surface of the RCR-complex and that mAb pairs binding to different antigens can function additively or synergistically to mediate parasite growth inhibition activity (GIA) in vitro. However, immunisation of rats with the RCR-complex consistently fails to outperform RH5.1 alone. We show this is due to immuno-dominance of RIPR coupled with the inferior potency of anti-full length RIPR polyclonal IgG antibodies as compared to the anti-RH5 and anti-CyRPA response. To address this, we identified the growth-inhibitory antibody epitopes of RIPR are clustered within C-terminal EGF-like domains of RIPR. A fusion of these EGF domains to CyRPA, called "R78C", combined with RH5.1, provided a new vaccination strategy that improves upon the levels of in vitro GIA seen with RH5.1 alone. Superiority of the combination antigen vaccine candidate was achieved by the induction of a quantitatively higher, but qualitatively similar, polyclonal antibody response that demonstrated additive GIA across the three antigen targets. These preclinical data justified the advancement of the RH5.1+R78C/Matrix-M combination vaccine to a Phase 1 clinical trial.

immunology↗

bioRxiv2024

Preclinical Development of a Stabilized RH5 Virus-Like Particle Vaccine that Induces Improved Anti-Malarial Antibodies

The development of a highly effective vaccine against the pathogenic blood-stage infection of human malaria will require a delivery platform that can induce an antibody response of both maximal quantity and functional quality. One strategy to achieve this includes presenting antigens to the immune system on virus-like particles (VLPs). Here we sought to improve the design and delivery of the blood-stage Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) antigen, which is currently in a Phase 2 clinical trial as a full-length soluble protein-in-adjuvant vaccine candidate called RH5.1/Matrix-M. We identify disordered regions of the full-length RH5 molecule induce non-growth inhibitory antibodies in human vaccinees, and a re-engineered and stabilized immunogen that includes just the alpha-helical core of RH5 induces a qualitatively superior growth-inhibitory antibody response in rats vaccinated with this protein formulated in Matrix-M adjuvant. In parallel, bioconjugation of this new immunogen, termed "RH5.2", to hepatitis B surface antigen VLPs using the "plug-and-display" SpyTag-SpyCatcher platform technology also enabled superior quantitative antibody immunogenicity over soluble antigen/adjuvant in vaccinated mice and rats. These studies identify a new blood-stage malaria vaccine candidate that may improve upon the current leading soluble protein vaccine candidate RH5.1/Matrix-M. The RH5.2-VLP/Matrix-M vaccine candidate is now under evaluation in Phase 1a/b clinical trials.

immunology↗

bioRxiv2023

Target-agnostic identification of human antibodies to Plasmodium falciparum sexual forms reveals cross stage recognition of glutamate-rich repeats

Circulating sexual stages of Plasmodium falciparum (Pf) can be transmitted from humans to mosquitoes, thereby furthering the spread of malaria in the population. It is well established that antibodies (Abs) can efficiently block parasite transmission. In search for naturally acquired Ab targets on sexual stages, we established an efficient method for target-agnostic single B cell activation followed by high-throughput selection of human monoclonal antibodies (mAbs) reactive to sexual stages of Pf in the form of gamete and gametocyte extract. We isolated mAbs reactive against a range of Pf proteins including well-established targets Pfs48/45 and Pfs230. One mAb, B1E11K, was cross-reactive to various proteins containing glutamate-rich repetitive elements expressed at different stages of the parasite life cycle. A crystal structure of two B1E11K Fab domains in complex with its main antigen, RESA, expressed on asexual blood stages, showed binding of B1E11K to a repeating epitope motif in a head-to-head conformation engaging in affinity-matured homotypic interactions. Thus, this mode of recognition of Pf proteins, previously described only for PfCSP, extends to other repeats expressed across various stages. The findings augment our understanding of immune-pathogen interactions to repeating elements of the Plasmodium parasite proteome and underscore the potential of the novel mAb identification method used to provide new insights into the natural humoral immune response against Pf. Impact StatementA naturally acquired human monoclonal antibody recognizes proteins expressed at different stages of the Plasmodium falciparum lifecycle through affinity-matured homotypic interactions with glutamate-rich repeats

immunology↗

bioRxiv2023

Analysis of the Diverse Antigenic Landscape of the Malaria Invasion Protein RH5 Identifies a Potent Vaccine-Induced Human Public Antibody Clonotype

The highly conserved and essential Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as the leading target for vaccines that seek to protect against the disease-causing blood-stage of malaria. However, the features of the human vaccine-induced antibody response that confer highly potent inhibition of malaria parasite invasion into red blood cells are not well defined. Here we characterize over 200 human IgG monoclonal antibodies induced by the most advanced PfRH5 vaccine. We define the antigenic landscape of this molecule, and establish epitope specificity, antibody association rate and intra-PfRH5 antibody interactions are key determinants of functional anti-parasitic potency. In addition, we identify a germline gene combination that results in an exceptionally potent class of antibody and demonstrate its prophylactic potential to protect against P. falciparum parasite challenge in vivo. This comprehensive dataset provides a framework to guide rational design of next-generation vaccines and prophylactic antibodies to protect against blood-stage malaria.

immunology↗

bioRxiv2023

Stochastic model of vesicular stomatitis virus replication reveals mutational effects on virion production

We present the first complete stochastic model of vesicular stomatitis virus (VSV) intracellular replication. Previous models developed to capture VSVs intracellular replication have either been ODE-based or have not represented the complete replicative cycle, limiting our ability to understand the impact of the stochastic nature of early cellular infections on virion production between cells and how these dynamics change in response to mutations. Our model accurately predicts changes in mean virion production in gene-shuffled VSV variants and can capture the distribution of the number of viruses produced. This model has allowed us to enhance our understanding of intercellular variability in virion production, which appears to be influenced by the duration of the early phase of infection, and variation between variants, arising from balancing the time the genome spends in the active state, the speed of incorporating new genomes into virions, and the production of viral components. Being a stochastic model, we can also assess other effects of mutations beyond just the mean number of virions produced, including the probability of aborted infections and the standard deviation of the number of virions produced. Our model provides a biologically interpretable framework for studying the stochastic nature of VSV replication, shedding light on the mechanisms underlying variation in virion production. In the future, this model could enable the design of more complex viral phenotypes when attenuating VSV, moving beyond solely considering the mean number of virions produced. Author SummaryThis study presents the first complete stochastic model of vesicular stomatitis virus (VSV) replication. Our model captures the dynamic process of VSVs replication within host cells, accounting for the stochastic nature of early cellular infections and how these dynamics change in response to mutations. By accurately predicting changes in mean virion production and the distribution of viruses in gene-shuffled VSV variants, our model enhances our understanding of viral replication and the variation we see in virion production. Importantly, our findings shed light on the mechanisms underlying the production of VSV virions, revealing the influence of factors such as the duration of the early infection phase and the interplay between the genomes ability to switch into an inactive state and viral protein production. We go beyond assessing the mean number of virions produced and examine other effects of mutations, including the probability of aborted infections and the variability in virion production. This stochastic model provides a valuable framework for studying the complex nature of viral replication, contributing to our understanding of single-cell viral dynamics and variability. Ultimately, this knowledge could pave the way for designing more effective strategies to attenuate VSV.

systems biology↗

bioRxiv2023

Pathogen-driven CRISPR screens identify TREX1 as a regulator of DNA self-sensing during influenza virus infection

Intracellular pathogens interact with host factors, exploiting those that enhance replication while countering those that suppress it. Genetic screens have begun to define the host:pathogen interface and establish a mechanistic basis for host-directed therapies. Yet, limitations of current approaches leave large regions of this interface unexplored. To uncover host factors with pro-pathogen functions, we developed a novel fitness-based screen that queries factors important during the middle-to-late stages of infection. This was achieved by engineering influenza virus to direct the screen by programing dCas9 to modulate host gene expression. A genome-wide screen identified the cytoplasmic DNA exonuclease TREX1 as a potent pro-viral factor. TREX1 normally degrades cytoplasmic DNA to prevent inappropriate innate immune activation by self DNA. Our mechanistic studies revealed that this same process functions during influenza virus infection to enhance replication. Infection triggered release of mitochondrial DNA into the cytoplasm, activating antiviral signaling via cGAS and STING. TREX1 metabolized the mitochondrial DNA preventing its sensing. Collectively, these data show that self-DNA is deployed to amplify host innate sensing during RNA virus infection, a process tempered by TREX1. Moreover, they demonstrate the power and generality of pathogen driven fitness-based screens to pinpoint key host regulators of intracellular pathogens.

microbiology↗

bioRxiv2022

Affinity-matured homotypic interactions induce spectrum of PfCSP-antibody structures that influence protection from malaria infection

The generation of high-quality antibody responses to PfCSP, the primary surface antigen of Plasmodium falciparum sporozoites, is paramount to the development of an effective malaria vaccine. Here we present an in-depth structural and functional analysis of a panel of potent antibodies encoded by the IGHV3-33 germline gene, which is among the most prevalent and potent antibody families induced in the anti-CSP immune response and targets the NANP repeat region. Cryo-EM reveals a remarkable spectrum of helical Fab-CSP structures stabilized by homotypic interactions between tightly packed Fabs, many of which correlate with somatic hypermutation. We demonstrate a key role of these mutated homotypic contacts for high avidity binding to CSP and in protection from P. falciparum malaria infection. These data emphasize the importance of anti-homotypic affinity maturation in the frequent selection of IGHV3-33 antibodies, advance our understanding of the mechanism(s) of antibody-mediated protection, and inform next generation CSP vaccine design.

immunology↗

bioRxiv2021

Vacuole phase-partitioning boosts mitochondria activity and cell lifespan through an inter-organelle lipid pipeline

Functional linkage between mitochondria and lysosomes is crucial for survival under starvation and lifespan extension. Despite such co-dependency, the supportive pathways connecting mitochondria and lysosomes remain unclear. Here, we identify an inter-organelle lipid trafficking pathway linking yeast vacuole and mitochondria that results in increased mitochondria growth and respiratory activity under glucose starvation. The pathway depends on vacuolar phase-separated, lipid domains, which provide zones for: activation of the vacuolar proton pump; lipid droplet (LD) docking and internalization; and, lipid transfer from vacuole-to-ER-to-mitochondria. Partitioned vacuolar domains form through a specialized type of macro-autophagy, triggered only under acute glucose starvation, that delivers sterol-rich, endosomal-derived lipids to the vacuole. To balance this lipid influx, the vacuole reroutes lipids back to the ER to support both LD biogenesis and mitochondria growth and activity. Energy produced by enhanced mitochondrial activity then feeds back to support the inter-organelle lipid trafficking pathways to ensure survival under nutrient stress.

cell biology↗

bioRxiv2020

Structural and biophysical correlation of anti-NANP antibodies with in vivo protection against P. falciparum

The most advanced P. falciparum circumsporozoite protein (PfCSP)-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the need to elicit more potent and durable antibody responses. Here, we elucidate crystal structures, binding affinities and kinetics, and in vivo protection of eight anti-NANP antibodies (Abs) derived from an RTS,S phase 2a trial and encoded by three different heavy-chain germline genes. The structures reinforce the importance of homotypic Fab-Fab interactions in protective Abs and the overwhelmingly dominant preference for a germline-encoded aromatic residue for recognition of the NANP motif. A number of biophysical properties were analyzed and antibody affinity correlated best with protection in an in vivo mouse model, with the more potent antibodies also recognizing epitopes with repeating secondary structural motifs of type I {beta}- and Asn pseudo 310 turns. Such insights can be incorporated into design of more effective immunogens as well as antibodies for passive immunization.

microbiology↗

bioRxiv2020

MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes

Mitochondrial and plastid genomes in land plants exhibit some of the slowest rates of sequence evolution observed in any eukaryotic genome, suggesting an exceptional ability to prevent or correct mutations. However, the mechanisms responsible for this extreme fidelity remain unclear. We tested seven candidate genes involved in cytoplasmic DNA replication, recombination, and repair (POLIA, POLIB, MSH1, RECA3, UNG, FPG, and OGG1) for effects on mutation rates in the model angiosperm Arabidopsis thaliana by applying a highly accurate DNA sequencing technique (duplex sequencing) that can detect newly arisen mitochondrial and plastid mutations still at low heteroplasmic frequencies. We find that disrupting MSH1 (but not the other candidate genes) leads to massive increases in the frequency of point mutations and small indels and changes to the mutation spectrum in mitochondrial and plastid DNA. We also used droplet digital PCR to show transmission of de novo heteroplasmies across generations in msh1 mutants, confirming a contribution to heritable mutation rates. This dual-targeted gene is part of an enigmatic lineage within the mutS mismatch repair family that we find is also present outside of green plants in multiple eukaryotic groups (stramenopiles, alveolates, haptophytes, and cryptomonads), as well as certain bacteria and viruses. MSH1 has previously been shown to limit ectopic recombination in plant cytoplasmic genomes. Our results point to a broader role in recognition and correction of errors in plant mitochondrial and plastid DNA sequence, leading to greatly suppressed mutation rates perhaps via initiation of double-stranded breaks and repair pathways based on faithful homologous recombination.

genetics↗

bioRxiv2018

Cryo-EM structure of the circumsporozoite protein of Plasmodium falciparum with a vaccine-elicited antibody reveals maturation of inter-antibody contacts

The circumsporozoite protein (CSP) on the surface of Plasmodium falciparum sporozoites is important for parasite development, motility, and host hepatocyte invasion. However, intrinsic disorder of the NANP repeat sequence in the central region of CSP has hindered its structural and functional characterization. Here, the cryo-EM structure at [~]3.4 [A] resolution of a recombinant shortened CSP construct (rsCSP) with the variable domains (Fabs) of a highly protective monoclonal antibody reveals an extended spiral conformation of the central NANP repeat region surrounded by antibodies. This unusual structure appears to be stabilized and/or induced by interaction with an antibody where contacts between adjacent Fabs are somatically mutated and enhance the interaction. Such maturation in non-antigen contact residues may be an effective mechanism for antibodies to target tandem repeat sequences and provide novel insights into malaria vaccine design.\n\nSummaryAn unusual spiral conformation is formed for the NANP repeat region in Plasmodium falciparum circumsporozoite protein (CSP) in complex with antibodies generated by the RTS,S vaccine and is stabilized by affinity-matured inter-Fab interactions.

immunology↗