Biology subjects

Dye, J.

Publications and source records attributed to Dye, J..

9 recordsLinked to original sources

bioRxiv2025

Rapid acquisition of HIV-1 neutralization breadth in a rhesus V2 apex germline antibody mouse model after a single bolus immunization

Current vaccine strategies to elicit broadly neutralizing antibodies (bnAbs) against HIV-1 generally propose complex, multi-boost immunization regimens. In rhesus macaques, SHIV infection has been observed to rapidly drive the development of some classes of bnAbs that share structural similarities with those in humans. Here, we generated a knockin mouse model with B cells bearing the unmutated common ancestor (UCA) of the V2 apex-targeted bnAb lineage, V033-a. A single immunization of mice with a germline-targeting native-like trimer was sufficient to recapitulate the ontogeny of the mature rhesus bnAb in knockin mice--including rare, disfavored somatic mutations--leading to the induction of antibodies that exhibited potent neutralization against both autologous and heterologous tier 2 viruses. A boost with Env escape mutant trimers further improved breadth and potency, and cryo-EM structure revealed the structural basis for heterologous neutralization breadth. Non-human primate and mouse models can thus combine with structure to serve as a platform for identifying and confirming immunogens that streamline HIV-vaccination regimens.

immunology↗

bioRxiv2025

Engineering gene expression dynamics via self-amplifying RNA with drug-responsive non-structural proteins

The design of gene therapies with drug-regulatable expression of therapeutic payloads is of interest for diverse applications. We hypothesized that a regulated expression system based on alphavirus-derived self-amplifying RNAs (saRNAs), which encode 4 non-structural proteins (nsPs) that copy the RNA backbone to enable sustained expression, would have advantages in safety and simplicity of delivery. Here we designed saRNAs where payload expression is regulated by the FDA-approved drug trimethoprim (TMP), by fusing TMP-responsive degradation domains (DDs) to nsPs to regulate RNA self-amplification. Screening a library of nsP-DD fusions, we identified an optimal design with DDs fused to nsP2, nsP3, and the payload, achieving a high fold-change in expression level in response to TMP and low expression in the off state. In mice, this saRNA circuit enabled diverse dynamic expression patterns in response to oral TMP. Implementing this circuit for controlled expression of an HIV antigen, an escalating TMP regimen significantly enhanced germinal center responses critical for B cell affinity maturation. This drug- regulated RNA technology holds potential for vaccines, immunotherapies, and gene therapies.

bioengineering↗

bioRxiv2025

Targeted suppression of type 1 interferon signaling during RNA delivery enhances vaccine-elicited immunity

RNA vaccines have emerged as a breakthrough technology, and one promising modality employs alphavirus-derived self-replicating RNA (repRNA) to express vaccine antigens. However, both the lipid nanoparticles (LNP) commonly used to deliver RNA and virus-like amplification of repRNAs trigger innate immune recognition, especially via type I interferon (IFN) signaling. To modulate IFN responses during vaccination, we formulated LNPs co-delivering antigen-encoding RNA together with siRNA targeting the interferon-/{beta} receptor-1 (IFNAR1). siRNA-mediated repression of IFNAR1 increased antigen expression from repRNAs by >10-fold, increased immune cell infiltration, and increased antigen presenting cell activation in the injection site and draining lymph nodes. Compared to repRNA alone, siRNA/repRNA co-delivery increased serum antibody titers >10-fold, dramatically augmented antigen-specific germinal center (GC) B cell responses, and primed 4.4-fold more antigen-specific T cells. Ifnar1 silencing by siRNA co-delivery similarly enhanced mRNA vaccines. Thus, siRNA co-delivery is a readily translatable approach to substantially enhance the immunogenicity of RNA vaccines.

bioengineering↗

bioRxiv2024

Visualizing lipid nanoparticle trafficking for mRNA vaccine delivery in non-human primates

mRNA delivered using lipid nanoparticles (LNPs) has become an important subunit vaccine modality, but mechanisms of action for mRNA vaccines remain incompletely understood. Here, we synthesized a metal chelator-lipid conjugate enabling positron emission tomography (PET) tracer labeling of LNP/mRNA vaccines for quantitative visualization of vaccine trafficking in live non-human primates (NHPs). Following i.m. injection, we observed LNPs distributing through injected muscle tissue, simultaneous with rapid trafficking to draining lymph nodes (dLNs). Deltoid injection of LNPs mimicking human vaccine administration led to stochastic LNP delivery to 3 different sets of dLNs. LNP uptake in dLNs was confirmed by histology, and cellular analysis of tissues via flow cytometry identified antigen-presenting cells as the primary cell type responsible for early LNP uptake and mRNA translation. These results provide insights into the biodistribution of mRNA vaccines administered at clinically relevant doses, injection volumes, and injection sites in an important large animal model for vaccine development.

bioengineering↗

bioRxiv2024

PIP4K2C inhibition reverses autophagic flux impairment induced by SARS-CoV-2

In search for broad-spectrum antivirals, we discovered a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrated selective dual inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Advanced lipidomics revealed alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and linked its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We discovered PIP4K2Cs roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays revealed that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced impairment of autophagic flux. Reversing this autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual inhibition of PIP4K2C and PIKfyve as a candidate strategy to combat emerging viruses.

microbiology↗

bioRxiv2021

Pan-ErbB inhibition protects from SARS-CoV-2 replication, inflammation, and injury

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

microbiology↗

bioRxiv2021

LY-CoV1404 potently neutralizes SARS-CoV-2 variants

SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization when administered early during COVID-19 disease. However, the emergence of variants of concern has negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolated a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody called LY-CoV1404 (also known as bebtelovimab). LY-CoV1404 potently neutralizes authentic SARS-CoV-2 virus, including the prototype, B.1.1.7, B.1.351 and B.1.617.2). In pseudovirus neutralization studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant and retains binding to spike proteins with a variety of underlying RBD mutations including K417N, L452R, E484K, and N501Y. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved with the exception of N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of reactivity to amino acid substitutions present among current VOC together with broad and potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants causing COVID-19. In BriefLY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated. HighlightsO_LILY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and known variants of concern including the B.1.1.529 (Omicron), the BA.2 Omicron subvariant, and B.1.617.2 (Delta) variants C_LIO_LINo loss of potency against currently circulating variants C_LIO_LIBinding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID database C_LIO_LIBreadth of neutralizing activity and potency supports clinical development C_LI

immunology↗

bioRxiv2020

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma

Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq1) to the spike ectodomain (S-ECD2) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro, we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 [A]2). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning3,4 our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.

immunology↗

bioRxiv2020

Relationship between Anti-Spike Protein Antibody Titers and SARS-CoV-2 In Vitro Virus Neutralization in Convalescent Plasma

Newly emerged pathogens such as SARS-CoV-2 highlight the urgent need for assays that detect levels of neutralizing antibodies that may be protective. We studied the relationship between anti-spike ectodomain (ECD) and anti-receptor binding domain (RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by two different in vitro assays using convalescent plasma samples obtained from 68 COVID-19 patients, including 13 who donated plasma multiple times. Only 23% (16/68) of donors had been hospitalized. We also studied 16 samples from subjects found to have anti-spike protein IgG during surveillance screening of asymptomatic individuals. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers, and in vitro VN titer. Anti-RBD plasma IgG correlated slightly better than anti-ECD IgG titer with VN titer. The probability of a VN titer [≥]160 was 80% or greater with anti-RBD or anti-ECD titers of [≥]1:1350. Thirty-seven percent (25/68) of convalescent plasma donors lacked VN titers [≥]160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease either VN or IgG titers. Analysis of 2,814 asymptomatic adults found 27 individuals with anti-RBD or anti-ECD IgG titers of [≥]1:1350, and evidence of VN [≥]1:160. Taken together, we conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titer of [≥]1:1350 may provide critical information about protection against COVID-19 disease.

immunology↗