Hector C. Aguilar

The continuous emergence of variants of concern (VoCs) represents a significant challenge to effectively control severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although FDA-approved vaccines and antivirals have been successfully developed and implemented for the prophylactic and therapeutic intervention of SARS-CoV-2 infection, recent VoCs could escape protection garnered by previous vaccine and antiviral approaches. Determining the efficacy of prophylactics and/or therapeutics against recent VoCs will assist in efficiently controlling currently circulating SARS-CoV-2 strains. We used our previously described bacterial artificial chromosome-based reverse genetics approach for Omicron BA.5 to generate a recombinant SARS-CoV-2 BA.5 encoding a fusion of ZsGreen to Nanoluciferase (rBA.5 ZsG-Nluc) from the locus of the viral nucleocapsid (N) protein separated by the porcine teschovirus-1 2A proteolytic cleavage site. The rBA.5 ZsG-Nluc replicates to levels comparable to recombinant BA.5 wild type (rBA.5 WT) and expresses high levels of ZsG and Nluc in cultured cells. This facilitates tracking viral infection and the identification of antivirals and neutralizing antibodies with EC ₅₀ and NT ₅₀ values, respectively, similar to those obtained with rBA.5 WT. Importantly, in Keratin-18 human angiotensin-converting enzyme-2 mice, rBA.5 ZsG-Nluc retains the same pathogenicity and ability to replicate in the lungs of infected mice as rBA.5 WT. Using rBA.5 ZsG-Nluc, we detected Nluc activity systemically and Nluc and ZsG expression in the lungs of infected mice using an in vivo imaging system. Our results demonstrate the feasibility of using rBA.5 ZsG-Nluc to track viral infections and identify prophylactics and therapeutics against recent SARS-CoV-2 VoCs in vitro , ex vivo , and in vivo . IMPORTANCE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of the coronavirus disease 2019 pandemic, is continually evolving to escape immunity acquired by previous natural infections or vaccinations. Moreover, recent SARS-CoV-2 variants of concern (VoCs) have acquired antiviral-resistant mutations to FDA-approved drugs. The emergence of these VoCs highlights the importance of identifying new prophylactics and therapeutics against currently circulating SARS-CoV-2 strains. We generated a recombinant bireporter Omicron BA.5 SARS-CoV-2 (rBA.5 ZsG-Nluc) that expresses reporter proteins, which are useful for cellular and whole animal studies, and has similar viral replication and pathogenicity to a wild-type recombinant Omicron BA.5 SARS-CoV-2. In Keratin-18 human angiotensin-converting enzyme-2 mice, rBA.5 ZsG-Nluc infection can be tracked systemically or in the lungs of infected mice using an in vivo imaging system. We establish a proof-of-concept platform of rBA.5 ZsG-Nluc in combination with an ancestral SARS-CoV-2 strain expressing mCherry to simultaneously identify antivirals and neutralizing antibodies against original and recent SARS-CoV-2 strains.

A substantial proportion of hospitalized COVID-19 patients require ICU admission, often associated with an imbalance between antiviral responses and inflammatory signaling leading to uncontrolled cytokine secretion. The SARS-CoV-2 nucleocapsid (N) protein is a known immune antagonist, but its role in macrophage-driven cytokine storms is unclear. We demonstrate that N functions in a stimulus-specific manner, specifically amplifying extracellular and dampening intracellular RNA sensing. Moreover, we show that this is a conserved feature of pathogenic betacoronaviruses through distinct mechanisms. Our interaction networks with SARS-CoV-2 variant N proteins suggest that the Delta variant N drives inflammation through interactions with several proteins, most notably, cGAS. Profiling of secreted cytokines revealed that N disrupts the secretome in a variant-specific manner. Most notably, we found that supernatants from the Delta variant N–expressing macrophages dramatically disrupt heart endothelial barriers, implicating N in COVID-19–associated cardiac complications. Our findings highlight N-mediated immune imbalance as a driver of severe COVID-19 and identify N as a promising therapeutic target to mitigate hyperinflammation.

Nipah virus (NiV) is a BSL-4 zoonotic paramyxovirus with ∼75% human mortality. The matrix protein (M) of NiV and other paramyxoviruses binds the inner leaflet of the cellular plasma membrane, orchestrating virion assembly by bringing together transmembrane glycoproteins (F/G) and ribonucleoprotein complexes (N). However, the interactions of these full-length proteins within membrane complexes remain elusive. Using cryo-electron tomography and subtomogram averaging of virus like particles (VLPs), we interrogated the protein:protein interactions of the main NiV structural proteins M/N/F/G. The M lattice structure determined to 7Å revealed a novel M-dimer arrangement that yielded two distinct repeating holes. Notably, F-trimers were arranged above only one of the two holes, dependent on F’s cytoplasmic tail. G was enriched in regions of higher M-VLP curvature, while N dramatically increased M-VLP pleomorphism. This work provides novel insights into paramyxoviral protein complexes, structures, and morphology.

Here we investigated the pathogenesis and contact transmission of bovine- and human-derived highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b genotype B3.13 viruses in mammalian models. Using reverse genetics, we rescued three naturally occurring viruses: rTX2/24 (bovine-derived), rTexas/37 and rMichigan/90 (both human-derived), and compared their infection dynamics, replication and pathogenicity with the wild-type bovine TX2/24 strain in vitro and in vivo . All four viruses demonstrated comparable replication kinetics in four mammalian cell lines. However, the rMichigan/90 strain exhibited significantly smaller plaques in bovine and human cells. In vivo studies showed that mice infected with any of the viruses succumbed to infection within 4-5 days; however, mice infected with the rMichigan/90 virus exhibited slightly lower viral replication and shedding compared to the other strains. Similarly, as in the mouse experiments, in hamsters, all viruses induced body weight loss and oral shedding, with robust virus replication observed in tissues, but the rMichigan/90 virus presented reduced replication and shedding. Contact transmission studies in hamsters revealed limited transmissibility for these viruses, with only one out of four animals inoculated with the rMichigan/90 virus transmitting it to a naïve contact. These findings indicate that both bovine- and human-derived H5N1 genotype B3.13 viruses present high pathogenicity in mammals, though the overall transmissibility remains low. Significance Statement: Influenza A H5N1 virus spilled over from wild birds into dairy cattle in the US in 2024. Since then, an increased number of human infections – with at least 71 confirmed cases – were confirmed. In the present study we evaluated and compared the pathogenicity and transmissibility of a bovine and two human H5N1 viruses of the genotype B3.13. Our results show that while all viruses are pathogenic in mouse and hamster models, the bovine isolate TX2/24 presents a broader tissue tropism than the human derived viruses. Contact transmission studies revealed a limited ability of these viruses to transmit in the hamster contact transmission model. These findings highlight the high pathogenicity of H5N1 viruses in mammals and demonstrate that that their transmissibility potential remains low.

Enveloped viruses are significant zoonotic disease threats with the potential to cause global pandemics. We identified a class of small-molecule sulfur-containing antiviral compounds (XM series) that broadly inhibit enveloped viruses. Multidisciplinary approaches revealed that XM compounds alter the viral membrane lipid chemical composition, enhance membrane order within the hydrophobic bilayer, and increase membrane phase transition temperatures. This mechanism inhibits membrane fusion and viral entry, while leaving the viral glycoproteins and genomes largely unaffected. Leveraging these unique properties, we develop a proof-of-concept whole inactivated influenza virus (IIV) vaccine using XM-01 (XM-01-IIV). In a mouse model, XM-01-IIV elicit significantly enhanced neutralizing antibody responses against hemagglutinin and neuraminidase compared to traditional paraformaldehyde-inactivated vaccines. Further, XM-01-IIV reduces morbidity and mortality following influenza challenge, achieving protection comparable to live virus vaccination. This promising class of broadly acting antivirals can be highly impactful in the development of highly potent inactivated vaccines for enveloped viruses.

Viral pandemics continue to threaten global health and economic stability. Despite medical advances, the absence of broad-spectrum antivirals (BSAs) prevents rapid responses to emerging viral threats. This is largely due to the lack of universal drug targets across diverse viral families and high variability among viral proteins. In this study, we evaluated 57 synthetic carbohydrate receptors (SCRs) for antiviral activity in cellulo using pseudotyped virus particles (PVPs) from six high-risk viruses across three families: Paramyxoviridae, Filoviridae, and Coronaviridae. Four SCRs inhibited all tested PVPs, and their efficacy was confirmed against live viruses including SARS-CoV-2, MERS-CoV, EBOV, MARV, NiV, and HeV. Notably, SCR005 and SCR007 , which exhibited minimal toxicity, significantly reduced SARS-CoV-2 infection in a severe animal model with a single dose. Mechanistic studies suggested that SCRs bind viral envelope N-glycans, blocking viral attachment and/or fusion. These results identify conserved viral N-glycans as promising BSA targets and establish SCRs as candidate prophylactic agents against enveloped viruses with pandemic potential.

The computational design of protein-based binders presents unique opportunities to access 'undruggable' targets, but effective binder design often relies on stable three-dimensional structures or structure-influenced latent spaces. Here we introduce PepMLM, a target sequence-conditioned designer of de novo linear peptide binders. Using a masking strategy that positions cognate peptide sequences at the C terminus of target protein sequences, PepMLM finetunes the ESM-2 protein language model to fully reconstruct the binder region, achieving low perplexities matching or improving upon validated peptide-protein sequence pairs. After successful in silico benchmarking with AlphaFold-based docking, we experimentally validate the efficacy of PepMLM through both binding and degradation assays. PepMLM-derived peptides demonstrate sequence-specific binding to cancer and reproductive targets, including NCAM1 and AMHR2, and enable targeted degradation of proteins across diverse disease contexts, from Huntington's disease to live viral infections. Altogether, PepMLM enables the design of candidate binders to any target protein, without requiring structural input, facilitating broad applications in therapeutic development.

Membrane proteins expressed on the surface of enveloped viruses are potent antigens in a vaccine, yet are difficult to produce and present due to their instability without a lipid scaffold. Current vaccination strategies that incorporate viral membrane proteins, such as live attenuated viruses, inactivated viruses, or extracellular vesicles, have limitations including lengthy production time, poor immunogenicity, extensive processing steps, and/or poor stability. Cell-free protein synthesis of viral membrane proteins offers a rapid, one-step method to assemble vaccine nanoparticles via cotranslational folding of membrane proteins into nanoscale liposomes. Here, we develop a vaccine candidate for the deadly Nipah virus (NiV), a highly lethal virus listed by the World Health Organization as a priority pathogen, by cell-free expressing two full-length Nipah virus membrane proteins. We demonstrate that both NiV fusion protein (NiV F) and NiV glycoprotein (NiV G) can be expressed and cotranslationally integrated into liposomes and that they fold into their native conformation. We find that the removal of a signal peptide sequence and the alteration of liposome lipid composition improve viral membrane protein incorporation. Furthermore, a lipid adjuvant, monophosphoryl lipid A (MPLA), can be readily added to liposomes without disrupting protein-vesicle loading or protein folding conformations. Finally, we demonstrate that our generated liposomal formulations lead to enhanced humoral responses in mice compared to empty and single-protein controls. This work establishes a platform to quickly assemble and present membrane antigens as multivalent vaccines that will enable a rapid response to the broad range of emerging pathogenic threats.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), triggered a global pandemic with profound social and economic consequences. The viral spike (S) protein has been identified as a key determinant of SARS-CoV-2 pathogenicity. In this study, we demonstrate that the Omicron BA.4 and BA.5 variants, which have closely related S proteins, exhibit different virulence in K18-hACE2 transgenic mice. A comparison of genomic sequences revealed key differences between variants BA.4 and BA.5, including a three amino acid deletion (ΔKSF) in the linker region of the non-structural protein 1 (NSP1) in BA.4. Using reverse genetic systems, we engineered a recombinant (r)SARS-CoV-2 BA.5 expressing BA.4 NSP1, which was significantly attenuated in vivo , similar to the natural BA.4 isolate, compared to rBA.5 wild-type (WT). This finding indicates that NSP1 is responsible, at least in part, for the differences in virulence between BA.4 and BA.5. Mechanistically, BA.4 NSP1 showed a reduced ability to inhibit host gene translation compared to BA.5 NSP1. Notably, a rSARS-CoV-2 WA1 original strain containing the same ΔKSF in NSP1 was also attenuated in vivo compared to rWA1 WT. Together, these findings highlight the contributions of the NSP1 linker region to inhibiting host gene expression and SARS-CoV-2 pathogenicity, as well as the feasibility of targeting NSP1 for the rational design of live-attenuated vaccines and/or antivirals. IMPORTANCE: Understanding why some SARS-CoV-2 variants cause more severe disease than others is crucial for proper management or for the rational design of prophylactic and therapeutic treatments. While most studies focus on the spike (S) protein, we found that another viral protein, NSP1, also plays a key role in disease severity among SARS-CoV-2 variants. A small deletion in NSP1, present in the Omicron BA.4 variant, weakens the virus ability to shut down the host’s immune response, making BA.4 less severe than BA.5. When we introduced the same deletion into the original SARS- CoV-2 WA1 strain, the virus also became less harmful. This discovery suggests that NSP1 is an important virulence factor and supports the feasibility of targeting NSP1 for the development of new prophylactic and therapeutic treatments against SARS-CoV-2. By uncovering NSP1’s role in pathogenesis, our study provides insights that could help in designing better strategies to combat future variants of SARS-CoV-2.

The COVID-19 pandemic has been driven by SARS-CoV-2 variants with enhanced transmission and immune escape. Apart from extensive evolution in the Spike protein, non-Spike mutations are accumulating across the entire viral genome and their functional impact is not well understood. To address the contribution of these mutations, we reconstructed genomes of recent Omicron variants with disabled Spike expression (replicons) to systematically compare their RNA replication capabilities independently from Spike. We also used a single reference replicon and complemented it with various Omicron variant Spike proteins to quantify viral entry capabilities in single-round infection assays. Viral entry and RNA replication were negatively correlated, suggesting that as variants evolve reduced entry functions under growing immune pressure on Spike, RNA replication increases as a compensatory mechanism. We identified multiple mutations across the viral genome that enhanced viral RNA replication. NSP6 emerged as a hotspot with a distinct L260F mutation independently arising in the BQ.1.1 and XBB.1.16 variants. Using mutant and revertant NSP6 viral clones, the L260F mutation was validated to enhance viral replication in cells and increase pathogenesis in mice. Notably, this mutation reduced host lipid droplet content by NSP6. Collectively, a systematic analysis of RNA replication of recent Omicron variants defined NSP6’s key role in viral RNA replication that provides insight into evolutionary trajectories of recent variants with possible therapeutic implications.