Biology subjects

Johnson, R. I.

Publications and source records attributed to Johnson, R. I..

5 recordsLinked to original sources

bioRxiv2023

The Dilute domain of Canoe is not essential for Canoe's role in linking adherens junctions to the cytoskeleton but contributes to robustness of morphogenesis

Robust linkage between cell-cell adherens junctions and the actomyosin cytoskeleton allows cells to change shape and move during morphogenesis without tearing tissues apart. The multidomain protein Drosophila Canoe and its mammalian homolog Afadin are critical for this linkage, and in their absence many events of morphogenesis fail. To define underlying mechanisms, we are taking Canoe apart, using Drosophila as our model. Canoe and Afadin share five folded protein domains, followed by a large intrinsically disordered region. The largest of these folded domains is the Dilute domain, which is found in Canoe/Afadin, their paralogs, and members of the MyosinV family. To define the roles of Canoes Dilute domain we have combined biochemical, genetic and cell biological assays. Use of the AlphaFold tools revealed the predicted structure of the Canoe/Afadin Dilute domain, providing similarities and contrasts with that of MyosinV. Our biochemical data suggest one potential shared function: the ability to dimerize. We next generated Drosophila mutants with the Dilute domain cleanly deleted. Surprisingly, these mutants are viable and fertile, and Canoe{Delta}DIL protein localizes to adherens junctions and is enriched at junctions under tension. However, when we reduce the dose of Canoe{Delta}DIL protein in a sensitized assay, it becomes clear it does not provide full wildtype function. Further, canoe{Delta}DIL mutants have defects in pupal eye development, another process that requires orchestrated cell rearrangements. Together, these data reveal the robustness in AJ-cytoskeletal connections during multiple embryonic and postembryonic events, and the power of natural selection to maintain protein structure even in robust systems.

cell biology↗

bioRxiv2021

Preclinical Efficacy of IMM-BCP-01, a Highly Active Patient-Derived Anti-SARS-CoV-2 Antibody Cocktail

Using an unbiased interrogation of the memory B cell repertoire of convalescent COVID-19 patients, we identified human antibodies that demonstrated robust antiviral activity in vitro and efficacy in vivo against all tested SARS-CoV-2 variants. Here, we describe the pre-clinical characterization of an antibody cocktail, IMM-BCP-01, that consists of three unique, patient-derived recombinant neutralizing antibodies directed at non-overlapping surfaces on the SARS-CoV-2 spike protein. Two antibodies, IMM20184 and IMM20190 directly block spike binding to the ACE2 receptor. Binding of the third antibody, IMM20253, to its unique epitope on the outer surface of RBD, alters the conformation of the spike trimer, promoting release of spike monomers. These antibodies decreased SARS-CoV-2 infection in the lungs of Syrian golden hamsters, and efficacy in vivo efficacy was associated with broad antiviral neutralizing activity against multiple SARS-CoV-2 variants and robust antiviral effector function response, including phagocytosis, ADCC, and complement pathway activation. Our pre-clinical data demonstrate that the three antibody cocktail IMM-BCP-01 shows promising potential for preventing or treating SARS-CoV-2 infection in susceptible individuals. One sentence summaryIMM-BCP-01 cocktail triggers Spike Trimer dissociation, neutralizes all tested variants in vitro, activates a robust effector response and dose-dependently inhibits virus in vivo.

immunology↗

bioRxiv2021

Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mice and non-human primates

The SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine. Here, two adeno-associated viral (AAV)-based vaccine candidates demonstrate potent immunogenicity in mouse and nonhuman primates following a single injection. Peak neutralizing antibody titers remain sustained at 5 months and are complemented by functional memory T-cells responses. The AAVrh32.33 capsid of the AAVCOVID vaccine is an engineered AAV to which no relevant pre-existing immunity exists in humans. Moreover, the vaccine is stable at room temperature for at least one month and is produced at high yields using established commercial manufacturing processes in the gene therapy industry. Thus, this methodology holds as a very promising single dose, thermostable vaccine platform well-suited to address emerging pathogens on a global scale.

immunology↗

bioRxiv2020

Molecular basis for a germline-biased neutralizing antibody response to SARS-CoV-2

The SARS-CoV-2 viral spike (S) protein mediates attachment and entry into host cells and is a major target of vaccine and drug design. Potent SARS-CoV-2 neutralizing antibodies derived from closely related antibody heavy chain genes (IGHV3-53 or 3-66) have been isolated from multiple COVID-19 convalescent individuals. These usually contain minimal somatic mutations and bind the S receptor-binding domain (RBD) to interfere with attachment to the cellular receptor angiotensin-converting enzyme 2 (ACE2). We used antigen-specific single B cell sorting to isolate S-reactive monoclonal antibodies from the blood of a COVID-19 convalescent individual. The seven most potent neutralizing antibodies were somatic variants of the same IGHV3-53-derived antibody and bind the RBD with varying affinity. We report X-ray crystal structures of four Fab variants bound to the RBD and use the structures to explain the basis for changes in RBD affinity. We show that a germline revertant antibody binds tightly to the SARS-CoV-2 RBD and neutralizes virus, and that gains in affinity for the RBD do not necessarily correlate with increased neutralization potency, suggesting that somatic mutation is not required to exert robust antiviral effect. Our studies clarify the molecular basis for a heavily germline-biased human antibody response to SARS-CoV-2.

microbiology↗

bioRxiv2020

A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent in vitro

Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase that forms a dimer and serves as the cellular receptor for SARS-CoV-2. It is also a key negative regulator of the renin-angiotensin system (RAS), conserved in mammals, which modulates vascular functions. We report here the properties of a trimeric ACE2 variant, created by a structure-based approach, with binding affinity of ~60 pM for the spike (S) protein of SARS-CoV-2, while preserving the wildtype peptidase activity as well as the ability to block activation of angiotensin II receptor type 1 in the RAS. Moreover, the engineered ACE2 potently inhibits infection of SARS-CoV-2 in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.

microbiology↗