Biology subjects

Langlois, R. A.

Publications and source records attributed to Langlois, R. A..

15 recordsLinked to original sources

bioRxiv2026

A mammalian fibroblast zoo reveals barriers to cross-species virus replication and differential regulation of antiviral immunity

Virus zoonosis represents a continual threat to humans. Viruses emerge in humans directly or indirectly through interactions with mammals. However, our understanding of virus-host susceptibility across the diversity of mammals is limited and experimental model systems are restricted to a very small set of model organisms. Mammals are highly diverse and different habitats, lifestyles, diets, and exposures to different pathogens have driven substantial genetic diversity. We generated a library of primary fibroblasts across 86 mammalian species and used these cells to build an atlas of virus-host susceptibility across a panel of arboviruses that infect diverse mammals including humans. Perturbing the innate immune system revealed some virus-host pairs where restriction is controlled by the immune system and others where there is an incompatibility with host factors. Transcriptomic profiling of the library in response to virus sensing mimicked by poly(I:C) or antiviral signaling with type I interferon reveals a core set of conserved genes that respond as well as substantial species- and order-specific responses. Together these data provide a comparative atlas of innate immune responses across mammals and offer a window into how these systems have evolved across animals.

immunology↗

bioRxiv2026

A chicken interferon stimulated gene screen reveals limited genes restricting influenza replication

Avian influenza A virus (IAV) is a major global health threat. Not only does it cause significant economic losses to the agricultural industry, but it also presents a risk of spillover into the human population. However, several obstacles must be overcome for a successful cross-species transmission event to occur. One of these obstacles is the innate immunity of the host. While the innate immune response to IAV in humans has been well-studied, the innate immune response to IAV in birds is not as well understood. Here, we explore the interferon-induced immune response by identifying interferon-stimulated genes (ISGs) in chicken, quail, and duck cells, and find that they are largely species-specific. Using this data, we performed a CRISPR knockout ISG screen against two strains of IAV, the mouse-adapted PR8, and the low pathogenicity avian IAV isolate WF10. We find that for PR8, IFITM3 is the dominant restrictor of viral replication with several additional hits with modest effects, consistent with previous work in mammals. For avian WF10, IFITM3 was also a hit however there were two additional hits absent for PR8, the HSP70 co-chaperone protein BAG5 and deubiquitinase OTUD4. Overall, these findings confirm the divergence of interferon-mediated immunity between species and establish a screening system that can be used to identify ISGs important for viral restriction in avian species. IMPORTANCEWild birds, especially waterfowl and shore birds, are the primary natural reservoir for influenza A viruses. These birds can transmit IAV to domestic poultry, where the spillover risk to humans increases. Chickens are the most common species of domestic poultry world-wide and are frequently infected with avian influenza viruses. Despite this, relatively little is known about how their immune system responds to influenza infection. This lack of knowledge creates a barrier to finding ways of preventing or mitigating IAV infection in chicken flocks. In this study, we characterized the interferon-induced immune response to IAV in chickens and found that only a small subset of ISGs were responsible for restricting IAV in chicken cells.

microbiology↗

bioRxiv2026

Dirty mice better recapitulate key features of mRNA vaccine immunogenicity observed in humans

Although specific pathogen free (SPF) mice have traditionally been used to test candidate vaccines, recent work has demonstrated that "dirty" mice with broad microbial exposure more appropriately recapitulate human immune responses. Using a model where lab mice are co-housed with pet store mice, we modeled SARS-CoV-2 mRNA vaccine responses in dirty and traditional SPF models. We found that dirty mice show reduced serum spike-binding antibody titers after prime and require a second booster dose to reach SPF-level spike antibody titers. Additionally, spike antibodies showed faster waning in dirty mice through 5 months post-vaccination, and neutralizing activity of these antibodies were reduced against Omicron variants, directly comparable with observations in humans. We further investigated the seasonality and consistency of pathogens in cohoused mice, and the impact of serial microbial exposure on our animal model system. We found that pathogen exposure and T cell activation remained consistent over time, and that a single co-housing event was sufficient to provide broad microbial exposure. This work demonstrates that the dirty mouse co-housing system is a promising, translationally representative approach to screen candidate mRNA vaccines for efficacy and durability prior to human clinical trials. Significance statementThe development of mRNA vaccines during the COVID-19 pandemic dramatically reduced hospitalization and death rates for infected individuals. However, booster vaccinations were required to achieve full efficacy, and protection waned over time. Our research leveraged a "dirty" mouse model to test whether SARS-CoV-2 mRNA vaccinations in animals with previous microbial exposure better modelled human immune responses. We found that dirty mice require a booster vaccination for full efficacy and experienced waning serum antibody titer over time. We propose this approach as a future model for robust preclinical mRNA vaccine testing.

immunology↗

bioRxiv2025

Intrinsic OASL expression licenses interferon induction during influenza A virus infection

Effective control of viral infection requires rapid induction of the innate immune response, especially the type I and type III interferon (IFN) systems. Despite the critical role of IFN induction in host defense, numerous studies have established that most cells fail to produce IFNs in response to viral stimuli. The specific factors that govern cellular heterogeneity in IFN induction potential during infection are not understood. To identify specific host factors that license some cells but not others to mount an IFN response to viral infection, we developed an approach for analyzing temporal scRNA-seq data of influenza A virus (IAV)-infected cells. This approach identified the expression of several interferon stimulated genes (ISGs) within pre-infection cells as correlates of IFN induction potential of those cells, post-infection. Validation experiments confirmed that intrinsic expression of the ISG OASL is essential for robust IFNL induction during IAV infection. Altogether, our findings reveal an important role for IFN-independent, intrinsic expression of ISGs in promoting IFN induction and provide new insights into the mechanisms that regulate cell-to-cell heterogeneity in innate immune activation.

immunology↗

bioRxiv2025

Endogenous antigen processing promotes mRNA vaccine CD4+ T cell responses

Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines elicit robust CD4+ T cell responses, which are essential for antiviral immunity1-3. While peptides presented to CD4+ T cells via major histocompatibility complex class II (MHC II) are traditionally thought to be derived from extracellular sources that are processed by antigen presenting cells (APCs) through the classical exogenous pathway4,5, the precise mechanisms of mRNA-LNP vaccine-specific CD4+ T cell priming remain unknown. Here, we investigated the role of alternative, endogenous antigen presentation pathways6,7 in inducing CD4+ T cell responses to mRNA-LNP vaccines. APCs treated with mRNA-LNP vaccines were consistently superior in activating T cells under conditions of endogenous, rather than exogenous, presentation. Immunization with an mRNA-LNP vaccine that excludes antigen expression in APCs resulted in lower antigen-specific CD4+ T cell, T follicular helper cell, and antibody responses than mice receiving control vaccine. In contrast, depletion of vaccine antigen from exogenous sources such as muscle cells resulted in little to no reduction in antigen-specific CD4+ T cells. Our findings demonstrate that direct presentation of endogenous antigen on MHC II is crucial to mRNA-LNP vaccine-induced immune responses and adds to a growing body of literature that redefines the paradigm of MHC II-restricted antigen processing and presentation.

immunology↗

bioRxiv2024

Mammalian ZAP and KHNYN independently restrict CpG-enriched avian viruses

Zoonotic viruses are an omnipresent threat to global health. Influenza A virus (IAV) transmits between birds, livestock, and humans. Proviral host factors involved in the cross-species interface are well known. Less is known about antiviral mechanisms that suppress IAV zoonoses. We observed CpG dinucleotide depletion in human IAV relative to avian IAV. Notably, human ZAP selectively depletes CpG-enriched viral RNAs with its cofactor KHNYN. ZAP is conserved in tetrapods but we uncovered that avian species lack KHNYN. We found that chicken ZAP may not affect IAV (PR8) or CpG enriched IAV (PR8CG). Human ZAP or KHNYN independently restricted CpG-enriched IAV PR8CG by overexpression in chicken cells and by combined knockout in human cells. Additionally, mammalian ZAP-L and KHNYN also independently restricted an avian retrovirus (ROSV). Curiously, platypus KHNYN, the most divergent from eutherian mammals, was also capable of independent restriction of multiple diverse viruses. We suggest that some mammalian KHNYN can act as a bona fide restriction factor with cell-autonomous activity. Furthermore, we speculate that through repeated contact between avian viruses and mammalian hosts, protein changes may accompany CpG-biased mutations or reassortment to evade mammalian ZAP and KHNYN.

microbiology↗

bioRxiv2024

High-throughput investigation of genetic design constraints in domesticated Influenza A Virus for transient gene delivery

Replication-incompetent single cycle infectious Influenza A Virus (sciIAV) has demonstrated utility as a research and vaccination platform. Protein-based therapeutics are increasingly attractive due to their high selectivity and potent efficacy but still suffer from low bioavailability and high manufacturing cost. Transient RNA-mediated delivery is a safe alternative that allows for expression of protein-based therapeutics within the target cells or tissues but is limited by delivery efficiency. Here, we develop recombinant sciIAV as a platform for transient gene delivery in vivo and in vitro for therapeutic, research, and manufacturing applications (in vivo antimicrobial production, cell culture contamination clearance, and production of antiviral proteins in vitro). While adapting the system to deliver new protein cargo we discovered expression differences presumably resulting from genetic context effects. We applied a high-throughput screen to map these within the 3'-untranslated and coding regions of the hemagglutinin-encoding segment 4. This screen revealed permissible mutations in the 3'-UTR and depletion of RNA level motifs in the N-terminal coding region.

bioengineering↗

bioRxiv2022

Primary infection with Zika virus provides one-way heterologous protection against Spondweni virus infection in rhesus macaques

Spondweni virus (SPONV) is the closest known relative of Zika virus (ZIKV). SPONV pathogenesis resembles that of ZIKV in pregnant mice, and both viruses are transmitted by Aedes aegypti mosquitoes. We aimed to develop a translational model to further understand SPONV transmission and pathogenesis. We found that cynomolgus macaques (Macaca fascicularis) inoculated with ZIKV or SPONV were susceptible to ZIKV, but resistant to SPONV infection. In contrast, rhesus macaques (Macaca mulatta) supported productive infection with both ZIKV and SPONV and developed robust neutralizing antibody responses. Crossover serial challenge in rhesus macaques revealed that SPONV immunity did not protect against ZIKV infection, whereas ZIKV immunity was fully protective against SPONV infection. These findings establish a viable model for future investigation into SPONV pathogenesis, and suggest the risk of SPONV emergence is low in areas with high ZIKV seroprevalence due to one-way cross-protection between ZIKV and SPONV. TeaserIdentification of asymmetric immune interactions between Zika and Spondweni viruses in macaque monkeys.

microbiology↗

bioRxiv2022

Influenza infection recruits distinct waves of regulatory T cells to the lung that limit lung resident IgA+ B cells

The role of regulatory T cells (Tregs) in limiting responses to pathogens in tissues remains poorly described. We used scRNA-Seq and a newly generated Foxp3-lineage reporter line (Foxp3-iDTR mice) to track Tregs in the lungs and peripheral blood following infection with influenza virus. Few Tregs of any type were found in the lung at steady-state. Following influenza infection Tregs expressing a strong interferon-stimulated gene signature (ISG-Tregs) appeared by day 3, peaked by day 7, and largely disappeared by day 21 post-infection. A second diverse wave of tissue-repair-like Tregs (TR-Tregs) appeared by day 10 and were maintained through day 21 post-infection. These two distinct Treg subsets had different gene expression patterns and distinct TCR repertoires. To establish the role of Tregs during influenza infection, we acutely ablated Tregs at day 6 post-infection; this resulted in a significant increase in IgA+ B cells in the lung. To determine whether distinct Tregs subsets could also be observed in response to respiratory viral infections in humans we analyzed scRNA-Seq datasets of patients with COVID-19. Peripheral blood from healthy human volunteers had multiple Treg subsets defined by unique gene expression patterns, but few ISG-Tregs. In contrast, two distinct Tregs subsets were expanded in COVID-19 patients - ISG-Tregs and IL32 expressing Tregs (16-fold and 2-fold increased, respectively). ISG-Tregs were present at significantly higher levels in patients with mild versus severe COVID-19, while IL32 expressing Tregs showed the opposite pattern. Thus, the Treg response to respiratory viruses in humans is also diverse and correlates with disease outcome.

immunology↗

bioRxiv2021

Influenza virus replication in cardiomyocytes drives heart dysfunction and fibrosis

Cardiac dysfunction is a common extrapulmonary complication of severe influenza virus infection. Prevailing models propose that influenza-associated heart dysfunction is indirectly triggered by cytokine mediated cardiotoxicity downstream of the inflamed lung, rather than by direct infection of cardiac tissue. To test the etiology of cardiac dysfunction resulting from influenza virus infection, we generated a novel recombinant H1N1 influenza A virus that was attenuated in cardiomyocytes by incorporation of target sequences for miRNAs expressed specifically in that cell type (miR133b and miR206). Compared with control virus, mice infected with the miR-targeted virus had significantly reduced heart viral titers, confirming cardiac attenuation of viral replication. The miR-targeted virus, however, was fully replicative and inflammatory in lungs when compared to control virus, and induced similar systemic weight loss. The miR-targeted virus induced considerably lower levels of cardiac arrhythmia, fibrosis, and inflammation, compared with control virus, in mice lacking interferon induced transmembrane protein 3 (IFITM3), which serve as the only available model for severe influenza-associated cardiac pathology. We conclude that robust replication of virus in the heart is required for pathology even when lung inflammation is severe. Indeed, we show that human stem cell-derived cardiomyocytes are susceptible to influenza virus infection. This work establishes a fundamental new paradigm in which influenza virus damages the heart through direct infection of cardiomyocytes.

immunology↗

bioRxiv2021

Nucleocapsid vaccine elicits spike-independent SARS-CoV-2 protective immunity

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing antibodies target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with HAd5 expressing the nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and k18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral antigens, even if they are not a target of neutralizing antibodies, to broaden epitope coverage and immune effector mechanisms.

immunology↗

bioRxiv2020

Single-cell RNA-sequencing reveals pervasive but highly cell type-specific genetic ancestry effects on the response to viral infection

Humans vary in their susceptibility to infectious disease, partly due to variation in the immune response following infection. Here, we used single-cell RNA-sequencing to quantify genetic contributions to this variation in peripheral blood mononuclear cells, focusing specifically on the transcriptional response to influenza infection. We find that monocytes are the most responsive to influenza infection, but that all cell types mount a conserved interferon response, which is stronger in individuals with increased European ancestry. By comparing European American and African American individuals, we show that genetic ancestry effects on expression are common, influencing 29% of genes, but highly cell type-specific. Further, we demonstrate that much of this population-associated expression variation is explained by cis expression quantitative trait loci, which are enriched for signatures of recent positive selection. Our findings establish common cis-regulatory variants--including those that are differentiated by genetic ancestry--as important determinants of the antiviral immune response.

genomics↗

bioRxiv2020

Single cell resolution of SARS-CoV-2 tropism, antiviral responses, and susceptibility to therapies in primary human airway epithelium

The human airway epithelium is the initial site of SARS-CoV-2 infection. We used flow cytometry and single cell RNA-sequencing to understand how the heterogeneity of this diverse cell population contributes to elements of viral tropism and pathogenesis, antiviral immunity, and treatment response to remdesivir. We found that, while a variety of epithelial cell types are susceptible to infection, ciliated cells are the predominant cell target of SARS-CoV-2. The host protease TMPRSS2 was required for infection of these cells. Importantly, remdesivir treatment effectively inhibited viral replication across cell types, and blunted hyperinflammatory responses. Induction of interferon responses within infected cells was rare and there was significant heterogeneity in the antiviral gene signatures, varying with the burden of infection in each cell. We also found that heavily infected secretory cells expressed abundant IL-6, a potential mediator of COVID-19 pathogenesis.

microbiology↗

bioRxiv2020

SARS-CoV-2 neutralization and serology testing of COVID-19 convalescent plasma from donors with non-severe disease

We determined the antigen binding activity of convalescent plasma units from 47 individuals with a history of non-severe COVID-19 using three clinical diagnostic serology assays (Beckman, DiaSorin, and Roche) with different SARS-CoV-2 targets. We compared these results with functional neutralization activity using a fluorescent reporter strain of SARS-CoV-2 in a microwell assay. This revealed positive correlations of varying strength (Spearman r = 0.37-0.52) between binding and neutralization. Donors age 48-75 had the highest neutralization activity. Units in the highest tertile of binding activity for each assay were enriched (75-82%) for those with the highest levels of neutralization.

immunology↗

bioRxiv2020

Analytical Validation of a COVID-19 qRT-PCR Detection Assay Using a 384-well Format and Three Extraction Methods

The COVID-19 global pandemic is an unprecedented health emergency. Insufficient access to testing has hampered effective public health interventions and patient care management in a number of countries. Furthermore, the availability of regulatory-cleared reagents has challenged widespread implementation of testing. We rapidly developed a qRT-PCR SARS-CoV-2 detection assay using a 384-well format and tested its analytic performance across multiple nucleic acid extraction kits. Our data shows robust analytic accuracy on residual clinical biospecimens. Limit of detection sensitivity and specificity was confirmed with currently available commercial reagents. Our methods and results provide valuable information for other high-complexity laboratories seeking to develop effective, local, laboratory-developed procedures with high-throughput capability to detect SARS-CoV-2.

pathology↗