Biology subjects

Frieman, M. B.

Publications and source records attributed to Frieman, M. B..

13 recordsLinked to original sources

bioRxiv2026

SARS-CoV-2 ORF8 modulates the upper respiratory tract inflammatory response to facilitate transmission

The ability of respiratory viruses to exploit host immune responses to promote transmission is a defining feature of pandemics. SARS-CoV-2 remains a major global public health threat because of its persistent evolution and capacity to counteract evolving immune defenses. Although the immune evasion properties of the SARS-CoV-2 Spike protein are well characterized, the contributions of other viral proteins to transmission remain poorly understood. Here, we used the infant mouse model to define the role of the accessory protein ORF8 in SARS-CoV-2 spread. We demonstrate that ORF8 supports efficient upper respiratory tract (URT) infection, infectious virus shedding, and host-to-host transmission. Mice infected with a recombinant SARS-CoV-2 strain lacking ORF8 (r{Delta}ORF8) had less infectious virus recovered from URT tissues and nasal secretions and transmitted less efficiently than mice infected with the isogenic ancestral strain rWA-1, which contains an intact ORF8. Recombinant viruses encoding naturally occurring ORF8 mutations exhibited distinct transmission phenotypes, with ORF8-deficient viruses resembling r{Delta}ORF8. Infection with ORF8-sufficient viruses induced greater macrophage recruitment, inflammatory cytokine production, and type-I interferon (IFN-I) signaling programs than ORF8-deficient viruses. Intranasal IFN{beta} supplementation partially restored URT shedding by r{Delta}ORF8-infected mice and rescued transmission to contacts, whereas blockade of the type I interferon receptor (IFNAR) in rWA-1-infected index mice reduced contact infection and transmission. Together, these findings demonstrate that ORF8 promotes SARS-CoV-2 transmission by engaging an IFN-I-associated inflammatory and secretory program in the URT that supports virus shedding from the infected host. These data identify ORF8 as a viral determinant of host mucosal responses that promote contagiousness. ImportanceEfficient host-to-host transmission underlies the success of respiratory viruses. Although SARS-CoV-2 research has largely focused on the Spike protein, accessory proteins can also shape viral fitness and spread. We previously identified ORF8 as a determinant of SARS-CoV-2 transmission. Here we show that ORF8 promotes SARS-CoV-2 infectious viral shedding and transmission by engaging IFN-I-associated inflammatory and secretory responses in the URT. These findings reveal how a SARS-CoV-2 accessory protein can exploit mucosal antiviral responses to increase host contagiousness.

microbiology↗

bioRxiv2026

Optimizing an avian influenza vaccine using a novel Bacterial Enzymatic Combinatorial Chemistry (BECC) TLR4 adjuvant

The development of broadly protective and dose-sparing influenza vaccines remains a critical challenge, particularly for zoonotic H5N1 strains with pandemic potential. This study evaluates BECC470s, a synthetic TLR4 adjuvant, for its ability to enhance the immunogenicity and protective efficacy of recombinant H5 hemagglutinin (rHA) vaccination in murine models. BECC470s-adjuvanted rHA elicited robust IgG1/IgG2a antibody responses and complete survival following homologous 2004 H5N1 challenge in a prime-boost model. Although BECC470s broadened antibody binding to both variable HA head and conserved stalk domains by ELISA, functional neutralizing antibody responses were restricted to the matched 2004 H5N1 isolate, with no detectable neutralization of H5N1 viruses isolated in 2022 or 2024. These data indicate that BECC470s enhances the magnitude and apparent breadth of binding antibody responses while maintaining strain-specific neutralizing activity, supporting its potential as an adjuvant for next-generation influenza vaccines while underscoring the need for further optimization to achieve true cross-neutralizing protection.

immunology↗

bioRxiv2025

A SARS-CoV-2 entry inhibitor trimerizes to lock the spike protein in a closed conformation

The SARS-CoV-2 spike protein binds its receptor ACE2 to initiate target-cell infection. To engage ACE2, at least one of the three receptor-binding domains (RBDs) of the spike must adopt the up orientation. Here we describe S22, a potent, bioavailable, and non-toxic inhibitor of BA.2 and all subsequent Omicron variants. Cryo-EM analyses showed that S22 assembled as a trimer in a previously uncharacterized pocket of the spike apex, stabilizing all three RBDs in the down orientation, thereby preventing ACE2 association. Binding studies, especially those using mixed S22-sensitive and -resistant spikes, imply a cooperative assembly of three S22 molecules with three RBDs, resulting in an unusually slow S22 off-rate. Consistent with its slow dissociation and favorable pharmacokinetics, S22 suppressed viral replication 100-fold in the lungs of XBB.1.5-infected mice. Thus, S22 potently inhibits Omicron entry through a distinct mechanism whereby a small compound assembles cooperatively as a trimer to stabilize spike in an inactive conformation.

microbiology↗

bioRxiv2025

Enhancing protective efficacy and immunogenicity of hemagglutinin-based influenza vaccine utilizing adjuvants developed by BECC

Seasonal influenza viruses continue to pose a significant threat, causing substantial morbidity and mortality in the US and worldwide despite the availability of vaccines and antivirals. These challenges may be addressed by improving vaccine immunogenicity through the inclusion of adjuvants that enhance immune responses against key antigens including influenza hemagglutinin (HA). BECC (Bacterial Enzymatic Combinatorial Chemistry) adjuvants are novel Toll-like Receptor 4 (TLR4) ligands created by modifying enzymes from lipid A synthesis pathways in Gram-negative bacteria. This study compares the ability of the biological and synthetic versions of these adjuvants to enhance the efficacy of recombinant HA (rHA) antigens in mouse influenza virus challenge. Mice immunized with rHA adjuvanted with BECCs stimulate the humoral and cell-mediated arms of the immune system without exhibiting cytotoxicity/pyrogenicity. A robust HA-specific immunoglobulin subtype, especially IgG2a, response was observed in mice adjuvanted with BECCs as compared to control adjuvants, MPL, and PHAD Further, animals adjuvanted with BECC470 cleared infection seven days post-infection, demonstrating their potential for further translational development. Vaccination adjuvanted with BECCs were also able to increase immune recognition of linear B and T cell epitopes when compared to control adjuvants, as well as induce durable immune response eighteen months post-vaccination. Together, these findings indicate that BECCs may serve as highly effective adjuvants in influenza vaccination. One Sentence Summary: Efficacy of engineered lipid A molecules as adjuvants for HA-based influenza vaccine

immunology↗

bioRxiv2024

The mammalian SKI complex is a broad-spectrum antiviral drug target that upregulates cellular cholesterol to inhibit viral replication

There is a need for the development of broad-spectrum antiviral compounds that can act as first line therapeutic countermeasures to emerging viral infections. Host-directed approaches present a promising avenue of development and carry the benefit of mitigating risks of viral escape mutants. We have previously found the SKI (super killer) complex to be a broad-spectrum, host-target with our lead compound ("UMB18") showing activity against influenza A virus, coronaviruses, and filoviruses. The SKI complex is a cytosolic RNA helicase and we previously found that UMB18 inhibited viral RNA production but did not further define the mechanism. Here, we demonstrate that UMB18 directly binds to SKIC8 of the SKI complex and through transcriptomic analysis of UMB18 treated A549 cells revealed an upregulation of genes in the mevalonate pathway which drives cholesterol synthesis. Further investigation validated the genetic upregulation and confirmed an increase in total cellular cholesterol. This upregulation was dependent on the sterol regulatory element binding proteins (SREBPs) and their regulator SCAP, the major regulators for cholesterol and fatty acid synthesis. Depletion of the SREBPs or SCAP with siRNA, or extraction of cholesterol with methyl {beta}-cyclodextrin attenuated UMB18 antiviral activity, emphasizing the role of increased cholesterol synthesis in this mechanism of action. Our findings further define the antiviral mechanism of a developmental host-directed therapeutic approach with broad applicability against emerging viral pathogens. Author SummaryThe COVID-19 pandemic has underscored the urgent need for effective countermeasures to novel and emerging viral pathogens. Our research presented here builds upon our previously published data on an experimental novel antiviral compound termed UMB18. We have found this compound capable of inhibiting replication of influenza A virus, coronaviruses and the filoviruses Marburg and Ebola virus, but did not fully define a mechanism of action. In this work, we demonstrate that UMB18 exerts antiviral activity by modulating cellular cholesterol levels. By targeting the SKI complex, UMB18 triggers an increase in endogenous cellular cholesterol which disrupts the fine balance viruses rely on for efficient infection. We demonstrate that this mechanism inhibits replication of SARS-CoV-2, revealing a previously undescribed host-directed strategy for antiviral intervention. These findings highlight UMB18s potential as a broad-spectrum antiviral agent and pave the way for further research into its mechanism and therapeutic applications, offering a promising avenue for development of antiviral countermeasures to current, novel and emerging pathogens.

microbiology↗

bioRxiv2022

Reduced SARS-CoV-2 mRNA vaccine immunogenicity and protection in mice with diet-induced obesity and insulin resistance.

BackgroundObesity and Type 2 Diabetes Mellitus (T2DM) are associated with an increased risk of severe outcomes from infectious diseases, including COVID-19. These conditions are also associated with distinct responses to immunization, including an impaired response to widely used SARS-CoV-2 mRNA vaccines. ObjectiveTo establish a connection between reduced immunization efficacy via modeling the effects of metabolic diseases on vaccine immunogenicity that is essential for the development of more effective vaccines for this distinct vulnerable population. MethodsWe utilized a murine model of diet-induced obesity and insulin resistance to model the effects of comorbid T2DM and obesity on vaccine immunogenicity and protection. ResultsMice fed a high-fat diet (HFD) developed obesity, hyperinsulinemia, and glucose intolerance. Relative to mice fed a normal diet (ND), HFD mice vaccinated with a SARS-CoV-2 mRNA vaccine exhibited significantly lower anti-spike IgG titers, predominantly in the IgG2c subclass, associated with a lower type 1 response, along with a 3.83-fold decrease in neutralizing titers. Furthermore, enhanced vaccine-induced spike-specific CD8+ T cell activation and protection from lung infection against SARS-CoV-2 challenge were seen only in ND mice but not in HFD mice. ConclusionWe demonstrate impaired immunity following SARS-CoV-2 mRNA immunization in a murine model of comorbid T2DM and obesity, supporting the need for further research into the basis for impaired anti-SARS-CoV-2 immunity in T2DM and investigation of novel approaches to enhance vaccine immunogenicity among those with metabolic diseases. Capsule summaryObesity and type 2 diabetes impair SARS-CoV-2 mRNA vaccine efficacy in a murine model.

immunology↗

bioRxiv2022

SIRT-1 connects autophagy and release of virus-containing vesicles during picornavirus infection

Enterovirus D68 is a re-emerging enterovirus that causes acute respiratory illness in infants and has recently been linked to Acute Flaccid Myelitis. Here, we show that the histone deacetylase, SIRT-1, is essential for autophagy and EV-D68 infection. Knockdown of SIRT-1 inhibits autophagy and reduces EV-D68 extracellular titers. The proviral activity of SIRT-1 does not require its deacetylase activity or functional autophagy. SIRT-1s proviral activity is, we demonstrate, mediated through the repression of ER stress. Inducing ER stress through thapsigargin treatment or SERCA2A knockdown in SIRT-1 knockdown cells had no additional effect on EV-D68 extracellular titers. Knockdown of SIRT-1 also decreases poliovirus and SARS-CoV-2 titers but not coxsackievirus B3. In non-lytic conditions, EV-D68 is primarily released in an enveloped form, and SIRT-1 is required for this process. Our data show that SIRT-1, through its translocation to the cytosol, is critical to promote the release of enveloped EV-D68 viral particles.

microbiology↗

bioRxiv2022

Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq

Genomic and proteomic screens have identified numerous host factors of SARS-CoV-2, but efficient delineation of their molecular roles during infection remains a challenge. Here we use Perturb-seq, combining genetic perturbations with a single-cell readout, to investigate how inactivation of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our high-dimensional data resolve complex phenotypes such as shifts in the stages of infection and modulations of the interferon response. However, only a small percentage of host factors showed such phenotypes upon perturbation. We further identified the NF-{kappa}B inhibitor I{kappa}B (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides massively parallel functional characterization of host factors of SARS-CoV-2 and quantitatively defines their roles both in virus-infected and bystander cells.

systems biology↗

bioRxiv2021

Phage-like particle vaccines are highly immunogenic and protect against pathogenic coronavirus infection and disease

The response by vaccine developers to the COVID-19 pandemic has been extraordinary with effective vaccines authorized for emergency use in the U.S. within one year of the appearance of the first COVID-19 cases. However, the emergence of SARS-CoV-2 variants and obstacles with the global rollout of new vaccines highlight the need for platforms that are amenable to rapid tuning and stable formulation to facilitate the logistics of vaccine delivery worldwide. We developed a "designer nanoparticle" platform using phage-like particles (PLPs) derived from bacteriophage lambda for multivalent display of antigens in rigorously defined ratios. Here, we engineered PLPs that display the receptor binding domain (RBD) protein from SARS-CoV-2 and MERS-CoV, alone (RBDSARS-PLPs, RBDMERS-PLPs) and in combination (hCoV-RBD PLPs). Functionalized particles possess physiochemical properties compatible with pharmaceutical standards and retain antigenicity. Following primary immunization, BALB/c mice immunized with RBDSARS- or RBDMERS-PLPs display serum RBD-specific IgG endpoint and live virus neutralization titers that, in the case of SARS-CoV-2, were comparable to those detected in convalescent plasma from infected patients. Further, these antibody levels remain elevated up to 6 months post-prime. In dose response studies, immunization with as little as one microgram of RBDSARS-PLPs elicited robust neutralizing antibody responses. Finally, animals immunized with RBDSARS-PLPs, RBDMERS-PLPs, and hCoV-RBD PLPs were protected against SARS-CoV-2 and/or MERS-CoV lung infection and disease. Collectively, these data suggest that the designer PLP system provides a platform for facile and rapid generation of single and multi-target vaccines.

immunology↗

bioRxiv2021

An adjuvanted SARS-CoV-2 RBD nanoparticle elicits neutralizing antibodies and fully protective immunity in aged mice

Development of affordable and effective vaccines that can also protect vulnerable populations such as the elderly from COVID-19-related morbidity and mortality is a public health priority. Here we took a systematic and iterative approach by testing several SARS-CoV-2 protein antigens and adjuvants to identify a combination that elicits neutralizing antibodies and protection in young and aged mice. In particular, SARS-CoV-2 receptorbinding domain (RBD) displayed as a protein nanoparticle (RBD-NP) was a highly effective antigen, and when formulated with an oil-in-water emulsion containing Carbohydrate fatty acid MonoSulphate derivative (CMS) induced the highest levels of cross-neutralizing antibodies compared to other oil-in-water emulsions or AS01B. Mechanistically, CMS induced antigen retention in the draining lymph node (dLN) and expression of cytokines, chemokines and type I interferon-stimulated genes at both injection site and dLN. Overall, CMS:RBD-NP is effective across multiple age groups and is an exemplar of a SARS-CoV-2 subunit vaccine tailored to the elderly.

immunology↗

bioRxiv2020

NVX-CoV2373 vaccine protects cynomolgus macaque upper and lower airways against SARS-CoV-2 challenge

There is an urgent need for a safe and protective vaccine to control the global spread of SARS-CoV-2 and prevent COVID-19. Here, we report the immunogenicity and protective efficacy of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length SARS-CoV-2 spike (S) glycoprotein stabilized in the prefusion conformation. Cynomolgus macaques (Macaca fascicularis) immunized with NVX-CoV2373 and the saponin-based Matrix-M adjuvant induced anti-S antibody that was neutralizing and blocked binding to the human angiotensin-converting enzyme 2 (hACE2) receptor. Following intranasal and intratracheal challenge with SARS-CoV-2, immunized macaques were protected against upper and lower infection and pulmonary disease. These results support ongoing phase 1/2 clinical studies of the safety and immunogenicity of NVX-CoV2327 vaccine (NCT04368988). HighlightsO_LIFull-length SARS-CoV-2 prefusion spike with Matrix-M1 (NVX-CoV2373) vaccine. C_LIO_LIInduced hACE2 receptor blocking and neutralizing antibodies in macaques. C_LIO_LIVaccine protected against SARS-CoV-2 replication in the nose and lungs. C_LIO_LIAbsence of pulmonary pathology in NVX-CoV2373 vaccinated macaques. C_LI

immunology↗

bioRxiv2020

SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 elicits immunogenicity in baboons and protection in mice

The COVID-19 pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd immunity to control the spread of SARS-CoV-2. Here, we report the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length spike (S) protein, stabilized in the prefusion conformation. Purified NVX-CoV2373 S form 27.2nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice and baboons, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicits high titer anti-S IgG that is associated with blockade of hACE2 receptor binding, virus neutralization, and protection against SARS-CoV-2 challenge in mice with no evidence of vaccine-associated enhanced respiratory disease (VAERD). NVX-CoV2373 vaccine also elicits multifunctional CD4+ and CD8+ T cells, CD4+ T follicular helper T cells (Tfh), and the generation of antigen-specific germinal center (GC) B cells in the spleen. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2327 with Matrix-M (NCT04368988).

microbiology↗

bioRxiv2018

Coronavirus S protein-induced fusion is blocked prior to hemifusion by Abl kinase inhibitors

Enveloped viruses gain entry into host cells by fusing with cellular membranes, a step required for virus replication. Coronaviruses, including the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and infectious bronchitis virus (IBV), fuse at the plasma membrane or use receptor-mediated endocytosis and fuse with endosomes depending on the cell or tissue type. The virus Spike (S) protein mediates fusion with the host cell membrane. We have shown previously that an Abl kinase inhibitor, imatinib, significantly reduces SARS-CoV and MERS-CoV viral titers and prevents endosomal entry by HIV SARS S and MERS S pseudotyped virions. SARS-CoV and MERS-CoV are classified as BSL-3 viruses, which can make experimentation into the cellular mechanisms involved in infection more challenging. Here, we use IBV, a BSL-2 virus, as a model for studying the role of Abl kinase activity during coronavirus infection. We found that imatinib and two specific Abl kinase inhibitors, GNF2 and GNF5, reduce IBV titers by blocking the first round of virus infection. Additionally, all three drugs prevented IBV S-induced syncytia formation prior to the hemifusion step. Our results indicate that membrane fusion (both virus-cell and cell-cell) is blocked in the presence of Abl kinase inhibitors. Studying the effects of Abl kinase inhibitors on IBV will be useful in identifying host cell pathways required for coronavirus infection. This will provide insight into possible therapeutic targets to treat infections by current as well as newly emerging coronaviruses.

microbiology↗