Biology subjects

Corbett-Helaire, K. S.

Publications and source records attributed to Corbett-Helaire, K. S..

7 recordsLinked to original sources

bioRxiv2026

Evaluating Spike Antigenicity across Endemic Human Coronavirus Models using Flow Virometry

While SARS-CoV-2 research has advanced rapidly since COVID-19, endemic human coronaviruses (HCoVs) remain comparatively understudied. Tools to phenotype spike (S), the primary antigenic target on coronaviruses, at the single-virion level could improve vaccine design by capturing variation in epitope availability and spike abundance. Here, we establish a calibrated flow virometry (FV) platform to quantify S antigenicity on native endemic (HCoV-229E, HCoV-OC43) and epidemic (SARS-CoV-2) coronaviruses directly in cell culture supernatants. FV revealed cell line-dependent differences in S antigenicity, including receptor-induced changes in epitope accessibility. Comparison of virion-associated S with recombinant stabilized S by ELISA and biolayer interferometry showed consistent binding for HCoV-OC43, MERS-CoV, and SARS-CoV-2, but differences for HCoV-229E, with FV resolving heterogeneity not captured by bulk assays. Finally, FV showed that HCoV-229E from patient-derived air-liquid interface cultures exhibited reduced antibody binding and distinct S antigenicity compared to cell line-derived virions. Together, these findings establish FV as a platform for single-virion analysis of HCoV antigenicity.

microbiology↗

bioRxiv2026

Evaluating the Utility of a Nanoscale Flow Cytometer for Detection of Surface Proteins on HIV and Extracellular Vesicles

BackgroundFlow virometry (FV) - the application of flow cytometry to viruses - has historically been hindered by the inability of cytometers to detect particles below [~]300 nm in size. However, advances in optics and fluidics have enabled cytometers primarily designed for cells to detect viruses and extracellular vesicles (EVs) through light scatter alone. In 2024, the CytoFLEX nano was released, marketed for the detection of particles as small as 40 nm; however, its performance has yet to be compared to a conventional instrument for FV. MethodsFV was utilized to evaluate performance of the CytoFLEX nano and a conventional flow cytometer (CytoFLEX S). Instrument scatter sensitivity was assessed using NIST beads (40-400 nm), and virus stocks [human immunodeficiency virus (HIV), human coronaviruses (HCoV)-229E and HCoV-OC43]. For fluorescence analysis, HIV virions were stained with PE- and BV421-conjugated antibodies targeting virion incorporated proteins (CD38, CD44), individually and in combination. Finally, HIV stocks were labeled with antibodies against the envelope (Env) glycoprotein and tetraspanins (CD9, CD81) to assess EVs within virus preparations. ResultsCompared to the CytoFLEX S, the CytoFLEX nano exhibited substantially greater scatter sensitivity, reflected by up to 50-fold higher signal-to-noise ratio across NIST-traceable beads and virus samples. This enabled clearer resolution of smaller populations, including bead populations < 70 nm that were undetectable on the CytoFLEX S, as well as improved resolution across all viruses. While both instruments reliably detected stained proteins on HIV virions, the CytoFLEX nano revealed a distinct population of tetraspanin-positive EVs within HIV stocks that was undetected on the CytoFLEX S. Using GFP-tagged HIV, we identified Env+ particles lacking GFP, indicating the presence of Env on EVs. ConclusionsThe CytoFLEX nano exhibited markedly improved scatter sensitivity compared to the CytoFLEX S, improving detection of viruses and enabling detection of EV populations that were undetectable on the conventional instrument. While both platforms performed similarly for surface protein labeling, additional consideration of spectral overlap was needed with the CytoFLEX nano in multicolor experiments. These findings highlight that the complementary strengths of each platform can be utilized to more comprehensively characterize virus and EV populations, providing new opportunities to investigate nanoparticle heterogeneity.

microbiology↗

bioRxiv2025

Unconventional linkers facilitate potent stabilized coronavirus stem antibody responses following nanoparticle vaccination

Vaccine technologies that protect against a range of related pathogens within viral families, such as human immunodeficiency virus (HIV), influenza, and coronaviruses (CoVs) represent the future of viral vaccine development. Towards developing broad-spectrum CoV and influenza vaccines, we and others previously designed and evaluated CoV and influenza stem antigens; but these elicited relatively weak and sub-neutralizing antibody (Ab) responses. Multivalent antigen display on nanoparticles (NPs) is an established strategy to enhance and shape immunogenicity. However, one facet of NP vaccines has been largely overlooked: the indispensable linker segment between the antigen and NP core. Here, we introduce de novo-designed rigid (L2) and rarely used long flexible (L6) linkers to optimally display antigens on NPs, target occluded epitopes, and enhance cross-reactive Ab responses, using prefusion-stabilized Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S-2P) and stem (SS) antigens as prototype antigens. Antigenic characterization of L2-NPs confirmed enhanced Ab binding and exposure of cross-reactive epitopes compared with L6-NPs and soluble antigens. Immunization with SS-L2-NPs elicited broader, more potent cross-reactive Ab responses across the seven human-infecting CoVs and pandemic threat WIV1-CoV, whereas SS-L6-NPs induced stronger neutralizing Ab responses against MERS-CoV, SARS-CoV-2, and WIV1-CoV. Ab competition and systems serology analyses revealed that SS-L2-NPs elicit robust Fc-mediated effector functions. By improving CoV-targeting Ab functionality, these linker approaches have the potential to confer broad-spectrum CoV protection and represent a promising strategy against hypervariable influenza and HIV viruses - as well as other broad viral families with pandemic potential.

bioengineering↗

bioRxiv2025

Gram-positive bacterial cell wall components inhibit herpes simplex virus infection.

The role of the mucosal microbiome in viral infections remains unclear. Genital herpes, caused by herpes simplex virus 1 and 2 (HSV-1 and HSV-2), is among the most prevalent sexually transmitted infections worldwide. Despite evidence linking vaginal Lactobacillus to protection against sexually transmitted viruses, the specific microbial components and mechanisms that mediate this defense are not well understood. Here, we show that multiple cell wall components from diverse gram-positive bacteria, including lactobacilli, inhibit HSV-1 and HSV-2 infection in cells and in a mouse model of genital herpes infection. Peptidoglycan (PG) and lipoteichoic acid (LTA), both major components of the gram-positive bacterial cell wall, significantly reduced HSV infectivity in vitro and improved survival and disease outcomes in mice. We further showed that Lactobacillus crispatus surface layer proteins SlpA and SlpB bind HSV-1 and inhibit infection. Antiviral effects of cell wall components were dose-dependent, relied on intact PG structure, and, in the case of PG and LTA, were independent of TLR2-mediated host signaling. Collectively, our findings identify a species-independent antiviral function for gram-positive bacterial cell wall components against HSV and suggest that the composition of the mucosal microbiome may play an underappreciated role in suppressing mucosal herpes infection in humans.

microbiology↗

bioRxiv2025

Human Coronavirus HKU1 Neutralizing Monoclonal Antibodies Target Diverse Epitopes Within and Around the TMPRSS2 Receptor Binding Site

Endemic human coronaviruses (HCoVs), such as HCoV-HKU1, account for [~]30% of common colds each year and can cause serious upper and lower respiratory infections, yet no licensed vaccines or therapeutic antibodies target HCoVs. Despite being endemic to the human population, little is known about the antigenic landscape of HCoV-HKU1. Here, we characterized key interactions between the HCoV-HKU1 spike (S) protein and monoclonal antibodies (mAbs) isolated from convalescent HCoV-HKU-1-positive peripheral blood mononuclear cells collected prior to the COVID-19 pandemic. We isolated 14 mAbs that bound distinct regions of S, including the receptor-binding domain (RBD), N-terminal domain, and S2 subunit. Structural and functional studies revealed three groups of RBD-specific mAbs targeting diverse footprints within and around the TMPRSS2 receptor-binding site: (1) H501-022, which recognizes the TMPRSS2-binding site and thereby blocks receptor engagement; (2) H501-008, which binds a distinct epitope outside the TMPRSS2-binding site that is shared with HCoV-OC43; and (3) H501-018, which recognizes both "up" and "down" RBD conformations at a distinct, non-overlapping epitope outside the TMPRSS2-binding site. H501-008 weakly neutralized live HCoV-OC43 in vitro and protected mice against lethal HCoV-OC43 challenge. Notably, the three RBD-specific mAbs potently neutralized HCoV-HKU1 pseudovirus in TMPRSS2-overexpressing cell lines, but exhibited limited neutralizing activity against authentic HCoV-HKU1 infection in primary human airway epithelial cells. Together, these findings structurally define the antigenic landscape within and around the HKU1 receptor-binding site and demonstrate that receptor-binding site recognition alone is insufficient to predict physiologically relevant antibody-mediated neutralization.

microbiology↗

bioRxiv2025

Rational Design of Multiclade Coronavirus Spike Immunodominant Domain Nanoparticles Elicit Broad Antibody Responses

Four seasonal endemic human coronaviruses (EhCoVs), HKU1-CoV, OC43-CoV, 229E-CoV, and NL63-CoV, are culprits of mild upper respiratory and periodic severe diseases in vulnerable populations. Despite their prevalence, understanding EhCoVs antigenic and immune signatures remains elusive. SARS-CoV-2 has evolved as the fifth EhCoV, requiring seasonal vaccination in most parts of the world, and currently, no other EhCoV vaccines are available. SARS-CoV-2 co-infection with EhCoVs increases disease severity; thus, combined vaccination may provide increased protection against seasonal EhCoVs overall. Here, we explored Spike (S) receptor binding domain (RBD) vs. N-terminal domain (NTD) B-cell immunodominance in EhCoV-positive convalescent donors and immunogenicity in mice. We found that while antibody and B-cell isotypes were relatively dominant to S NTD, mice immunized with S RBD elicited significantly higher binding and neutralizing antibody (nAb) responses. With that knowledge, we used computational methods to infer that EhCoV S sequences evolve into two main clades and designed chimeric immunodominant domains (IDDs) from both clades for each EhCoV. IDDs were scaffolded onto two-component nanoparticles (NPs) displaying each IDD separately (monovalent IDD NP); three {beta}-EhCoV IDDs (Mosaic-3 IDD NP); or five EhCoVs IDDs (Mosaic-5 IDD NP). Mice immunized with mosaic IDD NPs, but not soluble IDD antigens nor monovalent IDD NPs, elicited potent, broadly cross-reactive binding and neutralizing antibody (Ab) responses against SARS-CoV-2 variants, other EhCoVs, and Sarbecoviruses. System serology revealed that all four IDD immunogens elicited distinct Ab subclasses and Fc-effector functions, with mosaic-5 IDD NPs eliciting the most de novo Ab subclasses, distributions, and broader Fc-mediated immune mechanisms. Dissection of vaccine-immune sera revealed polyclonal Ab responses against multiple non-overlapping cross-reactive S epitopes. Due to elicitation of broad Ab responses with combinatory functionality, IDD NPs open new horizons for developing first-in-class supraseasonal EhCoV vaccine candidates, with potential to decrease frequent SARS-CoV-2 sequence updates and protect against other EhCoVs. Moreover, elicitation of Ab breadth that spans pandemic-threat Sarbecoviruses gives mosaic IDD NPs promise towards pandemic preparedness.

bioengineering↗

bioRxiv2024

Structural serology of polyclonal antibody responses to mRNA-1273 and NVX-CoV2373 COVID-19 vaccines

Current COVID-19 vaccines are largely limited in their ability to induce broad, durable immunity against emerging viral variants. Design and development of improved vaccines utilizing existing platforms requires an in-depth understanding of the antigenic and immunogenic properties of available vaccines. Here we examined the antigenicity of two of the original COVID-19 vaccines, mRNA-1273 and NVX-CoV2373, by electron microscopy-based polyclonal epitope mapping (EMPEM) of serum from immunized non-human primates (NHPs) and clinical trial donors. Both vaccines induce diverse polyclonal antibody (pAb) responses to the N-terminal domain (NTD) in addition to the receptor-binding domain (RBD) of the Spike protein, with the NTD supersite being an immunodominant epitope. High-resolution cryo-EMPEM studies revealed extensive pAb responses to and around the supersite with unique angles of approach and engagement. NTD supersite pAbs were also the most susceptible to variant mutations compared to other specificities, indicating that ongoing Spike ectodomain-based vaccine design strategies should consider immuno-masking this site to prevent induction of these strain-specific responses.

immunology↗