Peter J. Halfmann

Human infections with the highly pathogenic avian influenza viruses highlight the risk of novel pandemics, against which seasonal vaccines provide limited cross-protection. Whereas antibody responses to current vaccine candidates are typically biased toward the immunodominant and variable head region of hemagglutinin (HA), the stem region of HA is highly conserved and targeted by broadly neutralizing antibodies. Here we describe a reoriented H5 HA (reoH5HA _Phe , based on A/Vietnam/1203/2004) displayed on alum as a vaccine candidate to elicit stem-directed antibody responses and confer cross-protective immunity. We show that a single immunization with reoH5HA _Phe protected naïve mice against heterologous H5N1 lethal challenge (A/Texas/37/2024), and boosting with reoH5HA _Phe conferred complete heterologous protection in mice pre-immunized with seasonal vaccines. The stem-directed response elicited by reoH5HA _Phe also protected against heterosubtypic H1N1 challenge in both naïve and pre-immune mice. Electron microscopy polyclonal epitope mapping revealed that reoH5HA _Phe elicited antibodies targeting the central stem and anchor epitopes on heterologous H5 and H1 HAs, alongside antibodies against the (H5) vestigial esterase or (H1) lateral patch epitopes. Despite limited cross-neutralizing activity, increased stem-directed responses with balanced IgG1 and IgG2 subclasses reduced viral replication and improved survival. Our results highlight antigen reorientation as an effective approach for designing variant-resilient vaccines and demonstrate the potential of reoH5HA _Phe as an H5 pandemic influenza vaccine candidate that confers protective immunity against both emerging and seasonal influenza viruses. Significance Statement: The global spread and human infections of avian H5N1 influenza viruses raise concerns of novel pandemics, against which humans lack pre-existing immunity. Whereas current vaccine strategies, including mRNA vaccines, mostly protect against sequence-matched viruses, pandemic preparedness requires vaccine design that resists antigenic variation and viral evolution. Here we employ molecular control of antigen orientation to focus antibody responses toward evolutionarily conserved sites of influenza hemagglutinin. We designed a reoriented H5 hemagglutinin based on the historical sequence (A/Vietnam/1203/2004) and demonstrated robust protection against the contemporary virus (A/Texas/37/2024). Vaccinations with reoriented H5 hemagglutinin also conferred protection against historical and contemporary H1N1 viruses. Our approach has potential to provide protection against emerging, pandemic-potential viruses, while simultaneously boosting immunity to seasonal influenza viruses.

The ongoing emergence and reemergence of RNA viruses from multiple viral families highlights the urgent need for broad‐spectrum antiviral therapeutics. To address this issue, a series of 4′‐azido fleximer nucleoside analogs were synthesized and evaluated for broad‐spectrum antiviral activity against pathogens of epidemic/pandemic concern. One compound, KAD‐039, featuring a 2′, 3′‐diisobutyrate‐5′‐ProTide prodrug, exhibited potent antiviral activity across flaviviruses, filoviruses, and coronaviruses, with EC ₅₀ values ranging from 1 to 10 µM and minimal cytotoxicity (CC ₅₀ > 50 µM). Pharmacokinetic evaluation of KAD‐039 demonstrated high plasma stability in dog, monkey, and human plasma, with half‐lives ranging from 19.9 to 28 h but exhibited poor stability in human liver microsomes ( t _1/2 = 6.1 min). To help elucidate the mechanism of action of KAD‐039 against these viral families, computational molecular docking studies were performed using the expected triphosphate active form within the cap/GTP‐binding pockets of several viral methyltransferases and showed that KAD‐039TP docked into all of the viral MTases with similar or higher predicted affinity compared to the natural substrate. In addition, KAD‐039TP displayed reduced predicted affinity for the human N7 methyltransferase, suggesting low host toxicity. These results highlight 4′‐azido fleximer analogs as promising broad‐spectrum antivirals, potentially functioning through viral methyltransferase inhibition, and support further investigation.

The outbreak of clade 2.3.4.4b H5N1 viruses among U.S. dairy cattle has raised concerns that sustained circulation among agricultural mammals could facilitate viral adaptation toward efficient human transmission. However, the evolutionary dynamics governing such adaptation remain poorly understood. Here we investigated the evolution of two bovine-derived H5N1 B3.13 genotype viruses during infection and airborne transmission in ferrets, building on prior characterization of their robust replication and inefficient airborne transmission. Within hosts, viral genetic diversity was limited and viruses were subject to genetic drift and weak purifying selection. Transmission, when it occurred, was characterized by stringent bottlenecks that sharply reduced viral genetic diversity. We found no evidence of mammalian adaptation during infection or transmission. Together, these findings indicate that bovine-derived H5N1 viruses face evolutionary constraints during acute mammalian infection and transmission, limiting movement toward enhanced airborne spread. These constraints may help explain why efficient mammalian replication does not necessarily coincide with efficient transmission. Continued circulation of HA clade 2.3.4.4b viruses nevertheless creates repeated opportunities for rare but consequential evolutionary events, underscoring the importance of sustained surveillance and risk mitigation.

The genomic flexibility of orthocoronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is showcased by the presence of accessory genes, which vary in number among virus species and strains. Given this flexibility, the viral coding capacity can be artificially expanded to express a heterologous gene from the viral genome, thereby enabling the development of a viral vector platform. Here, we systematically explored the extra gene coding capacity of SARS-CoV-2 by inserting an extra reporter gene at every intergenic region in its genome. We revealed the entire scheme of its extra gene expression and identified a genomic location that stably expresses reporter genes while maintaining the wild-type viral phenotype. Using this construct, we developed a set of fluorescent and luminescent reporter SARS-CoV-2 viruses available for in vivo flow cytometry and in vitro antiviral screening. Flow cytometric analysis with these reporter viruses revealed cell type–specific dynamics of SARS-CoV-2 infection in the lung tissue of K18-hACE2 mice. Our findings offer a platform for SARS-CoV-2 genome engineering, providing a set of reporter viruses for research applications.

Ebola virus (EBOV) is likely a zoonotic and re-emerging virus that causes severe outbreaks of Ebola virus disease. The virus spreads to various tissues during the late stage of infection and has been detected in immune-privileged sites of survivors. However, the mechanism of how EBOV disseminates throughout the body is not completely elucidated. In this study, by using a biologically contained EBOVΔVP30 system, we demonstrate that a megakaryocytic-like MEG-01 cell line that stably expresses VP30 (MEG-01 VP30 cells) is susceptible to EBOVΔVP30 infection and that MEG-01 VP30 cells exposed to EBOVΔVP30 produce platelet-like particles (PLPs) that contain EBOV proteins and viral genetic material. We further found that the viral envelope glycoprotein is expressed on the surface of the produced PLPs and contributes to PLP internalization into recipient cells. In addition, viral mRNA and genome RNA are actively synthesized in these PLPs, which may lead to progeny EBOV production from recipient cells that internalize the PLPs. Taken together, our data provide new insights into the potential role of platelets in the widespread dissemination of EBOV and the pathogenesis of Ebola virus disease.

SARS-CoV-2 infection typically resolves within weeks, but rare cases of prolonged replication—sometimes exceeding a year—have been documented, particularly in immunocompromised individuals. These persistent infections pose health risks and may give rise to highly divergent variants, yet the underlying biology remains poorly understood. Here, we describe a model of SARS-CoV-2 persistence using transgenic Syrian hamsters (males) lacking the interleukin-2 receptor gamma subunit (IL2rg). Infection with the XBB.1.16 variant led to efficient viral replication in respiratory tissues by two weeks after infection, with dissemination to other sites, including the intestinal tract. Viral titers remained high in multiple tissues at 100 days after infection. Longitudinal oral swab sequencing revealed dynamic shifts in intrahost single-nucleotide variant (iSNV) frequencies, with constellations of iSNVs rising and falling together, consistent with strong genetic linkage. Synonymous and nonsynonymous mutations accumulated at similar rates, suggesting genetic drift as the dominant evolutionary force. Tissue- and swab-derived sequences revealed extensive within-host diversity and hinted at tissue-specific evolutionary trajectories. This model enables detailed investigation of SARS-CoV-2 persistence and within-host viral evolution and provides a controlled system to study how long-term replication in tissue reservoirs may contribute to viral diversification.

The S2 subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is highly conserved across coronavirus strains and therefore is a potential pan-coronavirus vaccine target. However, antibodies targeting this region are typically non-neutralizing. We report herein that S2-targeting antibodies from patients who recovered from SARS-CoV-2 infection bound only closely related sarbecovirus subgenus strains and, like most known S2 antibodies, none of these were neutralizing. In contrast, first-exposure, severe acutely infected COVID-19 patients predominantly induced back-boosted antibody-secreting cells imprinted against past common cold coronavirus strain OC43 that were cross-reactive to as many as five subgenera of betacoronavirus strains and gave rise to antibodies that were neutralizing and protective. The antibodies targeted two different sites: one defined by competition with stem helix antibodies, and the second to an underdescribed epitope at the apex of S2. These findings suggest that S2-targeted vaccines could strategically exploit controlled OC43 priming followed by SARS-CoV-2 boosting to enhance the breadth and quality of protective antibody responses.

To address the need for broadly protective SARS-CoV-2 vaccines, we developed an attenuated a SARS-CoV-2 vaccine virus that lacks the open reading frames of two viral structural proteins: the envelope (E) and membrane (M) proteins. This vaccine virus (ΔEM) replicates in a cell line stably expressing E and M but not in wild-type cells. Vaccination with ΔEM elicits a CD8 T-cell response against the viral spike and nucleocapsid proteins. Two vaccinations with ΔEM provide better protection of the lower respiratory tissues than a single dose against the Delta and Omicron XBB variants in hamsters. Moreover, ΔEM is effective as a booster in hamsters previously vaccinated with an mRNA-based vaccine, providing higher levels of protection in both respiratory tissues compared to the mRNA vaccine booster. Collectively, our data demonstrate the feasibility of a SARS-CoV-2 ΔEM vaccine candidate virus as a vaccine platform.

Chikungunya virus (CHIKV) replication relies on the multifunctional nsP2 protein, making it an attractive target for antiviral drug discovery. Here, we report the resolution of oxaspiropiperidine 1 , a first-in-class inhibitor of the CHIKV nsP2 RNA helicase (nsP2hel), into its constitutive enantiomers and characterization of their antiviral activity. The enantiomer ( R )- 1 exhibited potent inhibition of viral replication, nsP2hel ATPase activity, and dsRNA unwinding, while the ( S )- 1 enantiomer was >100-fold less active. The ( R )- 1 enantiomer also demonstrated high selectivity for CHIKV over other RNA viruses and for nsP2hel over other RNA helicases. Direct binding of ( R )- 1 to nsP2hel protein was confirmed by ¹⁹ F NMR. Biophysical and structural studies revealed conformational polymorphism in the spirocyclic scaffold of ( R )- 1 , suggesting a potential role of thermal mobility of the ligand in allosteric inhibition of nsP2hel. Collectively, these findings designate ( R )- 1 (RA-NSP2-1) as a high-quality chemical probe and ( S )- 1 (RA-NSP2-1N) as a negative control for probing the biology of alphavirus RNA helicases.