Biology subjects

Halfmann, P. J.

Publications and source records attributed to Halfmann, P. J..

11 recordsLinked to original sources

bioRxiv2026

Designing a reoriented H5 hemagglutinin to generate stem-directed, heterosubtypic immunity to influenza A viruses

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 (reoH5HAPhe, 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 reoH5HAPhe protected naive mice against heterologous H5N1 lethal challenge (A/Texas/37/2024), and boosting with reoH5HAPhe conferred complete heterologous protection in mice pre-immunized with seasonal vaccines. The stem-directed response elicited by reoH5HAPhe also protected against heterosubtypic H1N1 challenge in both naive and pre-immune mice. Electron microscopy polyclonal epitope mapping revealed that reoH5HAPhe 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 reoH5HAPhe as an H5 pandemic influenza vaccine candidate that confers protective immunity against both emerging and seasonal influenza viruses.

immunology↗

bioRxiv2026

A rapidly deployable CRISPR-Cas3 diagnostic platform for emerging RNA viruses

Rapidly converting viral genome information into deployable molecular tests remains a major challenge in outbreak preparedness. We developed CONAN-SWIFT (Simple Workflow for Isothermal Field Testing), a sequence-to-test platform that integrates computational assay design, reverse-transcription loop-mediated isothermal amplification, CRISPR-Cas3 detection, reagent lyophilization and lateral-flow readout. Sequence-guided assays for Andes virus and Bundibugyo virus were established within approximately three weeks and extended to four additional filoviruses. A web-based designer supported crRNA selection, and systematic RT-LAMP primer optimization improved amplification performance. Recombinant Escherichia coli-expressed Cascade enabled standardized preparation of lyophilized Cas3-detection reagents, which were combined with a battery-operated isothermal device. The portable system detected as few as 10 input RNA copies per reaction within approximately 40 min. It also detected viral RNA and biologically contained, replication-incompetent Ebola virus in spiked human blood and concentrated wastewater. These findings establish the analytical feasibility of a rapidly adaptable CRISPR-Cas3 engineering framework for decentralized detection of emerging RNA viruses.

bioengineering↗

bioRxiv2025

In Vivo Evolution of Monoclonal Antibody CR3022 to Achieve Cross-Neutralization of SARS-CoV-2 and Implications for Vaccine Strategies Against SARS-Related Viruses

The epitope that monoclonal CR3022 binds to represents a promising target for broad protection against a wide range of human and zoonotic coronaviruses. We developed a powerful model to evaluate antibody affinity maturation in vivo using immunoglobulin (Ig)-humanized mice that express the predicted germline heavy chain of antibody CR3022. SARS-CoV/SARS-CoV-2 sequential immunization led to the convergent evolution of the germline CR3022 through somatic hypermutation (SHM) that resembled the affinity-matured CR3022 from a human, but now also adapted to key variants and divergent sarbecoviruses. While simple prime-boost strategies drove CR3022-epitope targeting, an intensive vaccination protocol elicited dominant responses to other epitopes. X-ray crystal structures revealed that SARS-CoV-2-neutralizing CR3022-like antibodies exhibit enhanced affinity by increasing polar and electrostatic interactions. Overall, these findings show CR3022-like clones can be readily adapted through SHM to increase breadth and potency to sarbecoviruses by relatively minor shifts in affinity with appropriate vaccination strategies.

immunology↗

bioRxiv2022

Site of vulnerability on SARS-CoV-2 spike induces broadly protective antibody to antigenically distinct omicron SARS-CoV-2 subvariants

The rapid evolution of SARS-CoV-2 Omicron variants has emphasized the need to identify antibodies with broad neutralizing capabilities to inform future monoclonal therapies and vaccination strategies. Herein, we identify S728-1157, a broadly neutralizing antibody (bnAb) targeting the receptor-binding site (RBS) and derived from an individual previously infected with SARS-CoV-2 prior to the spread of variants of concern (VOCs). S728-1157 demonstrates broad cross-neutralization of all dominant variants including D614G, Beta, Delta, Kappa, Mu, and Omicron (BA.1/BA.2/BA.2.75/BA.4/BA.5/BL.1). Furthermore, it protected hamsters against in vivo challenges with wildtype, Delta, and BA.1 viruses. Structural analysis reveals that this antibody targets a class 1 epitope via multiple hydrophobic and polar interactions with its CDR-H3, in addition to common class 1 motifs in CDR-H1/CDR-H2. Importantly, this epitope is more readily accessible in the open and prefusion state, or in the hexaproline (6P)-stabilized spike constructs, as compared to diproline (2P) constructs. Overall, S728-1157 demonstrates broad therapeutic potential, and may inform target-driven vaccine design against future SARS-CoV-2 variants.

immunology↗

bioRxiv2022

Host network-based discovery of critical regulators of innate immunity, virus growth, and pathogenesis in influenza virus infection

Innate immunity is protective against viruses, but also can facilitate pathological infection responses. Despite intensive research, our understanding of the mechanisms that regulate innate immunity in virus infection remains incomplete. Systems biology-based data-driven modeling approaches hold substantial promise toward discovery of crucial innate immune signaling regulators, yet model-derived predictions are almost completely unexplored. Here, we carried out systematic experimental validation of candidate regulators predicted by a transcriptional association network model of influenza virus-infected cells. We identified dozens of novel innate immune signaling regulators with potent effects on the replication of influenza and other viruses, and importantly, we established the biological relevance of a validated regulator in vivo. Collectively, these findings aid in clarifying mechanisms of influenza virus pathogenicity and might lead to innovative approaches for treating influenza virus disease. Similar data-driven modeling strategies may be applicable for the study of other pathogen systems or immunological disorders.

microbiology↗

bioRxiv2022

Characterization of SARS-CoV-2 Omicron BA.2.75 clinical isolates

The prevalence of the Omicron subvariant BA.2.75 is rapidly increasing in India and Nepal. In addition, BA.2.75 has been detected in at least 34 other countries and is spreading globally. However, the virological features of BA.2.75 are largely unknown. Here, we evaluated the replicative ability and pathogenicity of BA.2.75 clinical isolates in Syrian hamsters. Although we found no substantial differences in weight change among hamsters infected with BA.2, BA.5, or BA.2.75, the replicative ability of BA.2.75 in the lungs was higher than that of BA.2 and BA.5. Of note, BA.2.75 caused focal viral pneumonia in hamsters, characterized by patchy inflammation interspersed in alveolar regions, which was not observed in BA.5-infected hamsters. Moreover, in competition assays, BA.2.75 replicated better than BA.5 in the lungs of hamsters. These results suggest that BA.2.75 can cause more severe respiratory disease than BA.5 and BA.2 and should be closely monitored.

microbiology↗

bioRxiv2022

Resilience of S309 and AZD7442 monoclonal antibody treatments against infection by SARS-CoV-2 Omicron lineage strains

Omicron variant strains encode large numbers of changes in the spike protein compared to historical SARS-CoV-2 isolates. Although in vitro studies have suggested that several monoclonal antibody therapies lose neutralizing activity against Omicron variants1-4, the effects in vivo remain largely unknown. Here, we report on the protective efficacy against three SARS-CoV-2 Omicron lineage strains (BA.1, BA.1.1, and BA.2) of two monoclonal antibody therapeutics (S309 [Vir Biotechnology] monotherapy and AZD7442 [AstraZeneca] combination), which correspond to ones used to treat or prevent SARS-CoV-2 infections in humans. Despite losses in neutralization potency in cell culture, S309 or AZD7442 treatments reduced BA.1, BA.1.1, and BA.2 lung infection in susceptible mice that express human ACE2 (K18-hACE2). Correlation analyses between in vitro neutralizing activity and reductions in viral burden in K18-hACE2 or human Fc{gamma} R transgenic mice suggest that S309 and AZD7442 have different mechanisms of protection against Omicron variants, with S309 utilizing Fc effector function interactions and AZD7442 acting principally by direct neutralization. Our data in mice demonstrate the resilience of S309 and AZD7442 mAbs against emerging SARS-CoV-2 variant strains and provide insight into the relationship between loss of antibody neutralization potency and retained protection in vivo.

microbiology↗

bioRxiv2022

Mapping SARS-CoV-2 antigenic relationships and serological responses

During the SARS-CoV-2 pandemic, multiple variants escaping pre-existing immunity emerged, causing concerns about continued protection. Here, we use antigenic cartography to analyze patterns of cross-reactivity among a panel of 21 variants and 15 groups of human sera obtained following primary infection with 10 different variants or after mRNA-1273 or mRNA-1273.351 vaccination. We find antigenic differences among pre-Omicron variants caused by substitutions at spike protein positions 417, 452, 484, and 501. Quantifying changes in response breadth over time and with additional vaccine doses, our results show the largest increase between 4 weeks and >3 months post-2nd dose. We find changes in immunodominance of different spike regions depending on the variant an individual was first exposed to, with implications for variant risk assessment and vaccine strain selection. One sentence summaryAntigenic Cartography of SARS-CoV-2 variants reveals amino acid substitutions governing immune escape and immunodominance patterns.

immunology↗

bioRxiv2022

Nasally-delivered interferon-{lambda} protects mice against upper and lower respiratory tract infection of SARS-CoV-2 variants including Omicron

Although vaccines and monoclonal antibody countermeasures have reduced the morbidity and mortality associated with SARS-CoV-2 infection, variants with constellations of mutations in the spike gene threaten their efficacy. Accordingly, antiviral interventions that are resistant to further virus evolution are needed. The host-derived cytokine IFN-{lambda} has been proposed as a possible treatment based on correlative studies in human COVID-19 patients. Here, we show IFN-{lambda} protects against SARS-CoV-2 B.1.351 (Beta) and B.1.1.529 (Omicron)variants in three strains of conventional and human ACE2 transgenic mice. Prophylaxis or therapy with nasally-delivered IFN-{lambda}2 limited infection of historical or variant (B.1.351 and B.1.1.529) SARS-CoV-2 strains in the upper and lower respiratory tracts without causing excessive inflammation. In the lung, IFN-{lambda} was produced preferentially in epithelial cells and acted on radio-resistant cells to protect against of SARS-CoV-2 infection. Thus, inhaled IFN-{lambda} may have promise as a treatment for evolving SARS-CoV-2 variants that develop resistance to antibody-based countermeasures.

microbiology↗

bioRxiv2020

Transmission of SARS-CoV-2 in domestic cats imposes a narrow bottleneck

The evolutionary mechanisms by which SARS-CoV-2 viruses adapt to mammalian hosts and, potentially, undergo antigenic evolution depend on the ways genetic variation is generated and selected within and between individual hosts. Using domestic cats as a model, we show that SARS-CoV-2 consensus sequences remain largely unchanged over time within hosts, while dynamic sub-consensus diversity reveals processes of genetic drift and weak purifying selection. We further identify a notable variant at amino acid position 655 in Spike (H655Y), which was previously shown to confer escape from human monoclonal antibodies. This variant arises rapidly and persists at intermediate frequencies in index cats. It also becomes fixed following transmission in two of three pairs. These dynamics suggest this site may be under positive selection in this system and illustrate how a variant can quickly arise and become fixed in parallel across multiple transmission pairs. Transmission of SARS-CoV-2 in cats involved a narrow bottleneck, with new infections founded by fewer than ten viruses. In RNA virus evolution, stochastic processes like narrow transmission bottlenecks and genetic drift typically act to constrain the overall pace of adaptive evolution. Our data suggest that here, positive selection in index cats followed by a narrow transmission bottleneck may have instead accelerated the fixation of S H655Y, a potentially beneficial SARS-CoV-2 variant. Overall, our study suggests species- and context-specific adaptations are likely to continue to emerge. This underscores the importance of continued genomic surveillance for new SARS-CoV-2 variants as well as heightened scrutiny for signatures of SARS-CoV-2 positive selection in humans and mammalian model systems. Author summaryThrough ongoing human adaptation, spill-back events from other animal intermediates, or with the distribution of vaccines and therapeutics, the landscape of SARS-CoV-2 genetic variation is certain to change. The evolutionary mechanisms by which SARS-CoV-2 will continue to adapt to mammalian hosts depend on genetic variation generated within and between hosts. Here, using domestic cats as a model, we show that within-host SARS-CoV-2 genetic variation is predominantly influenced by genetic drift and purifying selection. Transmission of SARS-CoV-2 between hosts is defined by a narrow transmission bottleneck, involving 2-5 viruses. We further identify a notable variant at amino acid position 655 in Spike (H655Y), which arises rapidly and is transmitted in cats. Spike H655Y has been previously shown to confer escape from human monoclonal antibodies and is currently found in over 1000 human sequences. Overall, our study suggests species- and context-specific adaptations are likely to continue to emerge, underscoring the importance of continued genomic surveillance in humans and non-human mammalian hosts.

microbiology↗

bioRxiv2020

Limited SARS-CoV-2 diversity within hosts and following passage in cell culture

Since the first reports of pneumonia associated with a novel coronavirus (COVID-19) emerged in Wuhan, Hubei province, China, there have been considerable efforts to sequence the causative virus, SARS-CoV-2 (also referred to as hCoV-19) and to make viral genomic information available quickly on shared repositories. As of 30 March 2020, 7,680 consensus sequences have been shared on GISAID, the principal repository for SARS-CoV-2 genetic information. These sequences are primarily consensus sequences from clinical and passaged samples, but few reports have looked at diversity of virus populations within individual hosts or cultures. Understanding such diversity is essential to understanding viral evolutionary dynamics. Here, we characterize within-host viral diversity from a primary isolate and passaged samples, all originally deriving from an individual returning from Wuhan, China, who was diagnosed with COVID-19 and subsequently sampled in Wisconsin, United States. We use a metagenomic approach with Oxford Nanopore Technologies (ONT) GridION in combination with Illumina MiSeq to capture minor within-host frequency variants [≥]1%. In a clinical swab obtained from the day of hospital presentation, we identify 15 single nucleotide variants (SNVs) [≥]1% frequency, primarily located in the largest gene - ORF1a. While viral diversity is low overall, the dominant genetic signatures are likely secondary to population size changes, with some evidence for mild purifying selection throughout the genome. We see little to no evidence for positive selection or ongoing adaptation of SARS-CoV-2 within cell culture or in the primary isolate evaluated in this study. Author SummaryWithin-host variants are critical for addressing molecular evolution questions, identifying selective pressures imposed by vaccine-induced immunity and antiviral therapeutics, and characterizing interhost dynamics, including the stringency and character of transmission bottlenecks. Here, we sequenced SARS-CoV-2 viruses isolated from a human host and from cell culture on three distinct Vero cell lines using Illumina and ONT technologies. We show that SARS-CoV-2 consensus sequences can remain stable through at least two serial passages on Vero 76 cells, suggesting SARS-CoV-2 can be propagated in cell culture in preparation for in-vitro and in-vivo studies without dramatic alterations of its genotype. However, we emphasize the need to deep-sequence viral stocks prior to use in experiments to characterize sub-consensus diversity that may alter outcomes.

microbiology↗