Biology subjects

Stone, E. T.

Publications and source records attributed to Stone, E. T..

8 recordsLinked to original sources

bioRxiv2025

mRNA-LNP Vaccines Encoding for Dengue Optimized prM/ENV Proteins Induce Protective Immunity without ADE

Dengue virus (DENV) is the most common mosquito-borne virus in the world, causing nearly 400 million infections annually, with the number of cases predict to increase over time. Despite this prevalence there are no widely approved vaccine for DENV naive individuals or therapeutics. DENV consists of four distinct serotypes, DENV 1-4, that share 60-70% homology. Development of a safe and efficacious pan-Dengue vaccine has been complicated by the potential of antibody-dependent enhancement, in which cross-reactive non-neutralizing antibodies can enhance infection and disease. We previously demonstrated that a mRNA-LNP encoding for DENV-1 prM and Envelope proteins generates homotypic neutralizing immune responses and protects against a lethal challenge. The DENV-1 vaccine avoided ADE by eliminating the fusion loop (FL), one of the dominant ADE epitopes. Using a similar design methodology, here we present mRNA-LNP prM-E vaccines for DENV 2, 3, and 4. These vaccines maintained the {Delta}FL modifications, but encode a chimeric E protein to improve VLP expression and stability. All three vaccines elicited neutralizing titers of serotype specific antibodies mice and protected against a lethal homotypic DENV challenge. Mutation of the FL lowered ADE in vaccinated mouse sera. We formulated monotypic vaccines into a tetravalent vaccine and evaluated for immunogenicity and protection in mice. The tetravalent vaccine elicited neutralizing humoral responses against all DENV serotypes and protected against lethal challenges. Cumulatively, our monovalent DENV mRNA-LNP vaccines against all DENV serotypes generate protective immunity and lower the potential for ADE, leading to the development of a first-generation ADE-altered tetravalent DENV vaccine.

microbiology↗

bioRxiv2025

High-resolution in situ structures of hantavirus glycoprotein tetramers

SUMMARY/ABSTRACTNew World hantaviruses cause severe infections in humans, with case fatality rates approaching 40%. Previous structural studies have advanced our understanding of hantavirus glycoprotein architecture and function, however, the lack of high-resolution in situ structures of the glycoprotein tetramer and its lattice organization has limited mechanistic insights into viral assembly, entry, and antigenicity. Here, we leveraged a virus-like particle (VLP) system to establish a cryo-electron microscopy workflow for lattice-forming viral glycoproteins. This enabled the determination of a 2.35 [A] resolution structure of the membrane-embedded Andes virus (ANDV) glycoprotein tetramer, as well as structures of dimers of tetramers and a complex with antibody ADI-65534. These structures reveal previously uncharacterized features of glycoprotein organization, stability, and pH-sensing. Immunization of mice with self-amplifying replicon RNA (repRNA) encoding ANDV-VLPs elicited high levels of glycoprotein-binding antibodies but equivalent titers of neutralizing antibodies compared to repRNA-encoded native ANDV glycoprotein complex. Collectively, these findings advance our understanding of hantavirus glycoprotein assemblies and their function, laying a foundation for structure-based vaccine design efforts.

microbiology↗

bioRxiv2025

Metabolic syndrome enhances SARS-CoV-2 disease severity and reduces mRNA vaccine efficacy in a mouse model

Metabolic syndrome (MetS) is a cluster of pathophysiological conditions linked to the disruption of metabolic processes associated with energy storage and consumption. Approximately one-third of adults in the United States are currently diagnosed with MetS. Patients with MetS experience higher mortality rates following SARS-CoV-2 infection and exhibit poor vaccine efficacy following influenza virus vaccination compared to metabolically healthy individuals. However, the specific impact of MetS on immune responses to SARS-CoV-2 infection and vaccination has not been widely studied. To address this gap, we utilized high-fat diet feeding to establish a murine model of MetS, in which mice exhibit the same diagnostic criteria for MetS as human patients. We then used high-fat diet-induced MetS mice and regular chow diet-fed wild-type mice to analyze immune responses to SARS-CoV-2 infection and vaccination. Following SARS-CoV-2 infection, we monitored mice for disease severity, measured levels of virally induced inflammation, and quantified viral titers in various tissues. Our results indicate that MetS mice exhibit accelerated mortality post-infection, accompanied by elevated mRNA levels of inflammatory cytokine transcripts at sites of infection. Additionally, MetS alters the degree of viral replication across various tissues. Furthermore, our vaccination studies revealed that MetS reduces the potency of vaccine-induced neutralizing antibodies against both the ancestral SARS-CoV-2 strain and the Delta variant. Overall, our findings suggest that MetS exacerbates SARS-CoV-2 disease severity and diminishes vaccine efficacy, underscoring the need for tailored strategies to protect individuals with MetS from severe outcomes following infection and vaccination. ImpactThis study represents the first detailed account of the failure to generate protective neutralizing antibody responses to SARS-CoV-2 in mice with metabolic syndrome (MetS) following vaccination. The insights gained from this research inform future vaccine design and aid in identifying individuals at greater risk of breakthrough infections. By specifically isolating the risk associated with MetS, rather than obesity alone, these findings lay the groundwork for future investigations aimed at enhancing immune responses in individuals with MetS. This work is highly relevant to a broad audience, as it addresses a critical unmet need in vaccine development and provides essential guidance for the rational design of vaccines to protect vulnerable populations affected by MetS.

immunology↗

bioRxiv2024

CD4+ and CD8+ T cells are required to prevent SARS-CoV-2 persistence in the nasal compartment

SARS-CoV-2 is the causative agent of COVID-19 and continues to pose a significant public health threat throughout the world. Following SARS-CoV-2 infection, virus-specific CD4+ and CD8+ T cells are rapidly generated to form effector and memory cells and persist in the blood for several months. However, the contribution of T cells in controlling SARS-CoV-2 infection within the respiratory tract are not well understood. Using C57BL/6 mice infected with a naturally occurring SARS-CoV-2 variant (B.1.351), we evaluated the role of T cells in the upper and lower respiratory tract. Following infection, SARS-CoV-2-specific CD4+ and CD8+ T cells are recruited to the respiratory tract and a vast proportion secrete the cytotoxic molecule Granzyme B. Using antibodies to deplete T cells prior to infection, we found that CD4+ and CD8+ T cells play distinct roles in the upper and lower respiratory tract. In the lungs, T cells play a minimal role in viral control with viral clearance occurring in the absence of both CD4+ and CD8+ T cells through 28 days post-infection. In the nasal compartment, depletion of both CD4+ and CD8+ T cells, but not individually, results in persistent and culturable virus replicating in the nasal compartment through 28 days post-infection. Using in situ hybridization, we found that SARS-CoV-2 infection persisted in the nasal epithelial layer of tandem CD4+ and CD8+ T cell-depleted mice. Sequence analysis of virus isolates from persistently infected mice revealed mutations spanning across the genome, including a deletion in ORF6. Overall, our findings highlight the importance of T cells in controlling virus replication within the respiratory tract during SARS-CoV-2 infection.

immunology↗

bioRxiv2022

Metal Coordinating Inhibitors of Rift Valley Fever virus Replication

Rift Valley Fever Virus (RVFV) is a veterinary and human pathogen and is an agent of bioterrorism concern. Currently, RVFV treatment is limited to supportive care, so new drugs to control RVFV infection are urgently needed. RVFV is a member of the Bunyavirales order, and replication of these viruses depends on the viral endonuclease activity of the viral L protein. Screening for RVFV replication inhibitors among compounds with divalent cation-coordinating motifs similar to known viral nuclease inhibitors identified 31 novel RVFV inhibitors with selective indexes from 5 - 402 and 50% effective concentrations of 0.54 - 56 {micro}M in Vero cells, primarily -Hydroxytropolones and N-Hydroxypyridinediones. Inhibitor activity and selective index was validated in the human cell line A549. To evaluate specificity, select compounds were tested against another Bunyavirus, La Crosse Virus (LACV). Conservation of the enzymatic activity such as the cap-snatching mechanism among the Bunyavirales implies that the -Hydroxytropolone and N-Hydroxypyridinedione chemotypes hold potential for development into treatments for related pathogens, including Hantaan Virus, Severe fever with thrombocytopenia syndrome virus, Crimean-Congo Hemorrhagic Fever Virus, and LACV. Keywords: Rift Valley Fever Virus 1, La Crosse virus 2, Cap-snatching endonuclease 3, Replication inhibitors 4, -Hydroxytropolones 5, N-Hydroxypyridinediones 6.

microbiology↗

bioRxiv2020

The receptor binding domain of SARS-CoV-2 spike is the key target of neutralizing antibody in human polyclonal sera.

Natural infection of SARS-CoV-2 in humans leads to the development of a strong neutralizing antibody response, however the immunodominant targets of the polyclonal neutralizing antibody response are still unknown. Here, we functionally define the role SARS-CoV-2 spike plays as a target of the human neutralizing antibody response. In this study, we identify the spike protein subunits that contain antigenic determinants and examine the neutralization capacity of polyclonal sera from a cohort of patients that tested qRT-PCR-positive for SARS-CoV-2. Using an ELISA format, we assessed binding of human sera to spike subunit 1 (S1), spike subunit 2 (S2) and the receptor binding domain (RBD) of spike. To functionally identify the key target of neutralizing antibody, we depleted sera of subunit-specific antibodies to determine the contribution of these individual subunits to the antigen-specific neutralizing antibody response. We show that epitopes within RBD are the target of a majority of the neutralizing antibodies in the human polyclonal antibody response. These data provide critical information for vaccine development and development of sensitive and specific serological testing.

immunology↗

bioRxiv2020

Characterization of cells susceptible to SARS-COV-2 and methods for detection of neutralizing antibody by focus forming assay

The SARS-CoV-2 outbreak and subsequent COVID-19 pandemic have highlighted the urgent need to determine what cells are susceptible to infection and for assays to detect and quantify SARS-CoV-2. Furthermore, the ongoing efforts for vaccine development have necessitated the development of rapid, high-throughput methods of quantifying infectious SARS-CoV-2, as well as the ability to screen human polyclonal sera samples for neutralizing antibodies against SARS-CoV-2. To this end, our lab has adapted focus forming assays for SARS-CoV-2 using Vero CCL-81 cells, referred to in this text as Vero WHO. Using the focus forming assay as the basis for screening cell susceptibility and to develop a focus reduction neutralization test. We have shown that this assay is a sensitive tool for determining SARS-CoV-2 neutralizing antibody titer in human, non-human primate, and mouse polyclonal sera following SARS-CoV-2 exposure. Additionally, we describe the viral growth kinetics of SARS-CoV-2 in a variety of different immortalized cell lines and demonstrate via human ACE2 and viral spike protein expression that these cell lines can support viral entry and replication.

pathology↗

bioRxiv2020

mRNA induced expression of human angiotensin-converting enzyme 2 in mice for the study of the adaptive immune response to severe acute respiratory syndrome coronavirus 2

The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic resulting in nearly 20 million infections across the globe, as of August 2020. Critical to the rapid evaluation of vaccines and antivirals is the development of tractable animal models of infection. The use of common laboratory strains of mice to this end is hindered by significant divergence of the angiotensin-converting enzyme 2 (ACE2), which is the receptor required for entry of SARS-CoV-2. In the current study, we designed and utilized an mRNA-based transfection system to induce expression of the hACE2 receptor in order to confer entry of SARS-CoV-2 in otherwise non-permissive cells. By employing this expression system in an in vivo setting, we were able to interrogate the adaptive immune response to SARS-CoV-2 in type 1 interferon receptor deficient mice. In doing so, we showed that the T cell response to SARS-CoV-2 is enhanced when hACE2 is expressed during infection. Moreover, we demonstrated that these responses are preserved in memory and are boosted upon secondary infection. Interestingly, we did not observe an enhancement of SARS-CoV-2 specific antibody responses with hACE2 induction. Importantly, using this system, we functionally identified the CD4+ and CD8+ peptide epitopes targeted during SARS-CoV-2 infection in H2b restricted mice. Antigen-specific CD8+ T cells in mice of this MHC haplotype primarily target peptides of the spike and membrane proteins, while the antigen-specific CD4+ T cells target peptides of the nucleocapsid, membrane, and spike proteins. The functional identification of these T cell epitopes will be critical for evaluation of vaccine efficacy in murine models of SARS-CoV-2. The use of this tractable expression system has the potential to be used in other instances of emerging infections in which the rapid development of an animal model is hindered by a lack of host susceptibility factors.

immunology↗