Biology subjects

Skinner, K.

Publications and source records attributed to Skinner, K..

7 recordsLinked to original sources

bioRxiv2025

Structural basis for synergistic antibody protection against the essential malaria invasion complex protein RIPR

Plasmodium falciparum RH5-interacting protein (RIPR) is central to the essential PTRAMP-CSS-RIPR-CyRPA-RH5 (PCRCR)-complex; a leading target of blood-stage malaria vaccines. However, mechanisms whereby anti-RIPR antibodies inhibit parasite invasion are poorly understood. Here, we characterise 83 human IgG mAbs from RIPR-vaccinated Kymouse platform mice. Single mAbs have minimal neutralising activity, however, high-level synergistic inhibition is observed with pools of mAbs targeting the RIPR-Tail region. Structural characterisation and molecular dynamics simulations of RIPR-Tail show that mAbs targeting EGF-like domains 6-8 (RIPREGF (6-8)), but not EGF-like domains 9-10 or the C-terminal domain (RIPREGF (9-10)-CTD), synergise to constrain the RIPR-Tail conformation. The same antibodies dissociate PTRAMP-CSS from RIPR, thereby enabling anti-RIPREGF (9-10)-CTD mAbs or anti-CSS sdAbs to bind and potentiate anti-RIPREGF (6-8) IgG. Addition of these mAbs to IgG from humans immunised with the R78C (RIPREGF (7-8)-CyRPA) candidate vaccine enhances malaria growth inhibition. These data provide a framework to guide next-generation blood-stage malaria vaccine design.

immunology↗

bioRxiv2024

Protein sequencing with single amino acid resolution discerns peptides that discriminate tropomyosin proteoforms

Protein variants of the same gene--proteoforms--can have high molecular similarity yet exhibit different biological functions. Thus, identifying unique peptides that unambiguously map to proteoforms can provide crucial biological insights. In humans, four human tropomyosin (TPM) genes produce similar proteoforms that can be challenging to distinguish with standard proteomics tools. For example, TPM1 and TPM2 share 85% sequence identity, with amino acid substitutions that play unique roles in muscle contraction and myopathies. In this study, we evaluated the ability of the recently released Platinum single-molecule protein sequencer to detect proteoform-informative peptides. Platinum employs fluorophore-labeled recognizers that reversibly bind to cognate N-terminal amino acids (NAAs), enabling polypeptide sequencing within nanoscale apertures of a semiconductor chip that can accommodate single peptide molecules. As a proof of concept, we evaluated the ability of Platinum to distinguish three main types of proteoform variation: paralog-level, transcript-level, and post-translational modification (PTM). We distinguished paralogous TPM1 and TPM2 peptides differing by a single isobaric residue (leucine/isoleucine). We also distinguished tissue-specific TPM2 spliceforms. Notably, we found that a phosphotyrosine-modified peptide displayed reduced recognizer affinity for tyrosine, showing sensitivity to PTMs. This study paves the way for the targeted detection of proteoform biomarkers at the single molecule level. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/621980v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@122c6eaorg.highwire.dtl.DTLVardef@315f7forg.highwire.dtl.DTLVardef@1748af2org.highwire.dtl.DTLVardef@4e2b21_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

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

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↗

bioRxiv2021

Human basigin (CD147) does not directly interact with SARS-CoV-2 spike glycoprotein

Basigin, or CD147, has been reported as a co-receptor used by SARS-CoV-2 to invade host cells. Basigin also has a well-established role in Plasmodium falciparum malaria infection of human erythrocytes where it is bound by one of the parasites invasion ligands, reticulocyte binding protein homolog 5 (RH5). Here, we sought to validate the claim that the receptor binding domain (RBD) of SARS-CoV-2 spike glycoprotein can form a complex with basigin, using RH5-basigin as a positive control. Using recombinantly expressed proteins, size exclusion chromatography and surface plasmon resonance, we show that neither RBD nor full-length spike glycoprotein bind to recombinant human basigin (either expressed in E. coli or mammalian cells). Given the immense interest in SARS-CoV-2 therapeutic targets, we would caution the inclusion of basigin in this list on the basis of its reported direct interaction with SARS-CoV-2 spike glycoprotein. ImportanceReducing the mortality and morbidity associated with COVID-19 remains a global health priority. Critical to these efforts is the identification of host factors that are essential to viral entry and replication. Basigin, or CD147, was previously identified as a possible therapeutic target based on the observation that it may act as a co-receptor for SARS-COV-2, binding to the receptor binding domain of the spike protein. Here, we show that there is no direct interaction between the RBD and basigin, casting doubt on its role as a co-receptor and plausibility as a therapeutic target.

biochemistry↗

bioRxiv2020

Wetland plant evolutionary history influences soil and endophyte microbial community composition

Methane is a microbially derived greenhouse gas whose emissions are highly variable throughout wetland ecosystems. Differences in plant community composition account for some of this variability, suggesting an influence of plant species on microbial community structure and function in these ecosystems. Given that closely related plant species have similar morphological and biochemical features, we hypothesize that plant evolutionary history is related to differences in microbial community composition. To examine species-specific patterns in microbiomes, we selected five monoculture-forming wetland plant species based on the evolutionary distances among them. We detected significant differences in microbial communities between sample types (unvegetated soil, bulk soil, rhizosphere soil, internal root tissues, and internal leaf tissues) associated with these plant species based on 16S relative abundances. We additionally found that differences in plant evolutionary history were correlated with variation in microbial communities across plant species within each sample type. Using qPCR, we observed substantial differences in overall methanogen and methanotroph population sizes between plant species and sample types. Methanogens tended to be most abundant in rhizosphere soils while methanotrophs were the most abundant in roots. Given that microbes influence methane flux and that plants affect methanogen and methanotroph populations, plant species contribute to variable degrees of methane emissions. Incorporating the influence of plant evolutionary history into future modeling efforts may improve predictions of wetland methane emission since microbial community differences correlate with differences in plant evolutionary history.

ecology↗