Biology subjects

Stanfield, R.

Publications and source records attributed to Stanfield, R..

5 recordsLinked to original sources

bioRxiv2026

PIP2;1 aquaporin promotes early stomatal closure in grapevine leaves during water stress

Aquaporins play a key role in plant responses to drought. Our previous work showed limited embolism in grapevine leaves under mild water stress and suggested that the outside-xylem water pathway plays a dominant role in reducing leaf hydraulic conductance (Kleaf) during dehydration. We used CRISPR-Cas9 to knockout the PIP2;1 aquaporin encoding gene in Vitis vinifera cv. Chardonnay to study how leaf function during dehydration is affected by this aquaporin isoform. We measured functional responses like stomatal and photosynthetic responses as well as Kleaf to compare wild-type and two independent PIP2;1 knockout lines. Under moderate drought, mutants maintained greater stomatal conductance (gs) and photosynthetic rates as {Psi}w declined. No significant differences were observed in mesophyll conductance (gm) across genotypes, however, mutants exhibited slightly higher values under moderate drought. Interestingly, all lines exhibited similar Kleaf vulnerabilities to drought. Our findings show that PIP2;1 induces earlier stomatal closure during dehydration while not modulating Kleaf responses across genotypes. This rapid response in WT plants would prevent further water loss that would lead to higher xylem tensions that can lead to embolism. These findings show that multiple mechanisms collectively limit leaf gas exchange and water loss during dehydration, enhancing our understanding of plant resilience to changing environments.

plant biology↗

bioRxiv2025

Structural and genetic signatures of two classes of HCV E2 neutralizing face antibodies from non-human primates immunized with a recombinant E1E2

Hepatitis C continues to be a significant public health problem despite advancements in antiviral therapeutics. To eliminate this disease, an effective vaccine against new infections and re-infections is needed. However, to date only one Hepatitis C virus (HCV) envelope protein (E1E2) immunogen, developed by Chiron Inc., has been tested in a Phase I clinical trial (ClinicalTrials.gov identifier NCT00500747). To establish a benchmark for elicitation of broadly neutralizing antibodies (bnAbs) by E1E2, we previously immunized non-human primates (NHPs) with this immunogen and isolated monoclonal nAbs that exhibit neutralization potency comparable to human nAbs. Here we show that NHP nAbs, encoded by germline genes IGHV1-138*01 and IGHV4-NL_5*01 (homologs of human IGHV1-69*10 and IGHV4-59*12, respectively), recognize a relatively E2 conserved region (neutralizing face) proximal to antigenic region 3 (AR3). These NHP AR3-targeting nAbs share highly similar binding modes to human AR3-targeting nAbs, suggesting a similarity in human and NHP immune responses to the same HCV immunogen.

immunology↗

bioRxiv2025

Rational design of next-generation filovirus vaccines with glycoprotein stabilization, nanoparticle display, and glycan modification

Filoviruses pose a significant threat to human health due to frequent outbreaks and high mortality. Although two vector-based vaccines are available for Ebola virus, a broadly protective filovirus vaccine remains elusive. Here, we evaluate a general strategy for stabilizing glycoprotein (GP) structures from Ebola, Sudan, and Bundibugyo orthoebolaviruses and Ravn orthomarburgvirus. A 3.2 [A] crystal structure provides atomic-level details of the redesigned Ebola virus GP, while cryo-electron microscopy reveals how a pan-orthoebolavirus neutralizing antibody targets a conserved site on the stabilized Sudan virus GP (3.13 [A] resolution), along with a low-resolution model of antibody-bound Ravn virus GP. A self-assembling protein nanoparticle (SApNP), I3-01v9, is redesigned at the N terminus to enable optimal surface display of filovirus GP trimers. Following detailed in vitro characterization, we examine the lymph node dynamics of Sudan virus GP and GP-presenting SApNPs in mice. Compared with the soluble trimer, SApNPs exhibit [~]112-fold longer retention in lymph node follicles, up to 28-fold greater presentation on follicular dendritic cell dendrites, and up to 3-fold stronger germinal center reactions. Functional antibody responses induced by filovirus GP trimers and SApNPs bearing wild-type and modified glycans are assessed in mice. This study provides a foundation for next-generation filovirus vaccine development. ONE-SENTENCE SUMMARYFilovirus glycoproteins and nanoparticles were rationally designed and characterized in vitro and in vivo to aid filovirus vaccine development.

microbiology↗

bioRxiv2024

Immunization of cows with HIV envelope trimers generates broadly neutralizing antibodies to the V2-apex from the ultralong CDRH3 repertoire

The generation of broadly neutralizing antibodies (bnAbs) to specific HIV epitopes of the HIV Envelope (Env) is one of the cornerstones of HIV vaccine research. The current animal models we use have been unable to reliable produce a broadly neutralizing antibody response, with the exception of cows. Cows have rapidly and reliably produced a CD4 binding site response by homologous prime and boosting with a native-like Env trimer. In small animal models other engineered immunogens previously have been able to focus antibody responses to the bnAb V2-apex region of Env. Here, we immunized two groups of cows (n=4) with two regiments of V2-apex focusing immunogens to investigate whether antibody responses could be directed to the V2-apex on Env. Group 1 were immunized with chimpanzee simian immunodeficiency virus (SIV)-Env trimer that shares its V2-apex with HIV, followed by immunization with C108, a V2-apex focusing immunogen, and finally boosted with a cross-clade native-like trimer cocktail. Group 2 were immunized with HIV C108 Env trimer followed by the same HIV trimer cocktail as Group 1. Longitudinal serum analysis showed that one cow in each group developed serum neutralizing antibody responses to the V2-apex. Eight and 11 bnAbs were isolated from Group 1 and Group 2 cows respectively. The best bnAbs had both medium breadth and potency. Potent and broad responses developed later than previous CD4bs cow bnAbs and required several different immunogens. All isolated bnAbs were derived from the ultralong CDRH3 repertoire. The finding that cow antibodies can target multiple broadly neutralizing epitopes on the HIV surface reveals important insight into the generation of immunogens and testing in the cow animal model. The exclusive isolation of ultralong CDRH3 bnAbs, despite only comprising a small percent of the cow repertoire, suggests these antibodies outcompete the long and short CDRH3 antibodies during the bnAb response. Author SummaryThe elicitation of epitope-specific broadly neutralizing antibodies is highly desirable for an HIV vaccine as bnAbs can prevent HIV infection in robust animal challenge models and humans, but to date, cows are the only model shown to reliably produce HIV bnAb responses on Envelope (Env) immunization. These responses involve Abs with ultralong CDRH3s and are all directed to a single site, the CD4 binding site. To determine whether this is a unique phenomenon or whether cow antibodies can target further bnAb sites on Env, we employed an immunization protocol that generated cow bnAbs to a second site, the V2-apex. We conclude that ultralong CDRH3s are well adapted to penetrate the glycan shield of HIV Env and recognize conserved regions and may constitute protein units, either in the context of antibodies or in other engineered proteins, that could be deployed as anti-HIV reagents.

immunology↗

bioRxiv2020

Single-component multilayered self-assembling nanoparticles presenting rationally designed glycoprotein trimers as Ebola virus vaccines

Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1C) regions to GP metastability. Specific stalk and HR1C modifications in a mucin-deleted form (GP{Delta}muc) increase trimer yield, whereas alterations of HR1C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GP{Delta}muc trimers in their unliganded state. We then display a modified GP{Delta}muc trimer on reengineered nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display.

microbiology↗