Biology subjects

Richner, J. M.

Publications and source records attributed to Richner, J. M..

12 recordsLinked to original sources

bioRxiv2026

Salivary microRNA Profiling of Long COVID Subjects Reveals Host-Encoded Regulators of Inflammation and Viral Persistence

Periodontal disease and COVID-19 are linked by convergent immunoinflammatory pathways, yet the molecular basis of their interaction remains poorly defined. Here, we present a comprehensive salivary microRNA profile from individuals with prior SARS-CoV-2 infection, sampled approximately 3-6 months after diagnosis and meeting criteria for long COVID, providing new insight into the post-viral oral microenvironment. Salivary miRNA sequencing revealed widespread repression in patients with PD, consistent with persistent immune dysregulation. Relative to COVID-19-negative/PD-negative controls, thirty-two miRNAs were differentially expressed in COVID-19-positive/PD-positive individuals, all significantly downregulated. A similar signature was observed in a post-vaccination cohort for the selected dysregulated miRNAs. Integrative pathway analyses identified these miRNAs as regulators of core inflammatory circuits, including Ras, MAPK, and NF{kappa}B signaling, converging on IL-1{beta}- and TNF-centered networks relevant to both PD and COVID-19. Mechanistically, restoration of three downregulated miRNAs, miR- miR-30e-3p 106-3p-3p, and miR-652-3p attenuated NF{kappa}B activation and cytokine release in TLR-stimulated human oral keratinocytes, while their functional suppression using inhibitors potentiates inflammation. These miRNAs were also predicted to target SARS-CoV-2 spike and nucleocapsid transcripts, an interaction validated by dual-luciferase reporter assays. Their overexpression further reduced spike and nucleocapsid expression in Beta- and Omicron-infected epithelial cells, as measured by flow cytometry and RT-qPCR confirming host miRNAs as potent endogenous SARS-CoV-2 restriction factor. Together, these findings identify salivary host miRNAs as mechanistic regulators of oral inflammatory tone and viral persistence, establishing a molecular link between periodontal inflammation and post-COVID oral pathology.

immunology↗

bioRxiv2025

Persistent Immune Dysregulation during Post-Acute Sequelae of COVID-19 is Manifested in Antibodies Targeting Envelope and Nucleocapsid Proteins

Post-Acute Sequelae of SARS-CoV-2 infection (PASC) syndrome or "Long COVID" represents a widespread health challenge that necessitates the development of novel diagnostic approaches and targeted therapies that can be readily deployed. Immune dysregulation has been reported as one of the hallmarks of PASC, but the extent of PASC immune dysregulation in patients over time remains unclear. We therefore assessed SARS-CoV-2-specific antibody responses, peripheral immune cell profiles, autoantibody profiles and circulating cytokines for up to 6 months in participants with a SARS-CoV-2 infection who either convalesced or developed PASC. Compared to convalescent, PASC participants with a broad range of PASC phenotypes exhibited persistently elevated IgG titers for SARS-CoV-2 Envelope and Nucleocapsid proteins over the 6 months of study duration. In contrast, the IgG responses to Spike protein were significantly lower in the PASC cohort with predominantly IgG1 and IgG3 class-switched bias. Using CyTOF analysis, we show elevated numbers of circulating T follicular helper cells (cTFH) and mucosa-associated invariant T cells (MAIT), which also correlated with high anti-Envelope IgG titers. Persistent immune activation was accompanied by augmented serum cytokine profiles with LIF, IL-11, Eotaxin-3, and HMGB-1 in PASC participants, who also demonstrated significantly higher rates of autoantibodies. These findings highlight the persistence of immune dysregulation in PASC, underscoring the need to explore targeted therapies addressing viral persistence, dysregulated antibody production, and autoimmunity.

immunology↗

bioRxiv2025

mRNA-LNP Vaccines Encoding for Dengue Optimized prM/ENV Proteins Induce Protective Immunity without ADE

Dengue virus (DENV) is the most common mosquito-borne virus in the world, causing nearly 400 million infections annually, with the number of cases predict to increase over time. Despite this prevalence there are no widely approved vaccine for DENV naive individuals or therapeutics. DENV consists of four distinct serotypes, DENV 1-4, that share 60-70% homology. Development of a safe and efficacious pan-Dengue vaccine has been complicated by the potential of antibody-dependent enhancement, in which cross-reactive non-neutralizing antibodies can enhance infection and disease. We previously demonstrated that a mRNA-LNP encoding for DENV-1 prM and Envelope proteins generates homotypic neutralizing immune responses and protects against a lethal challenge. The DENV-1 vaccine avoided ADE by eliminating the fusion loop (FL), one of the dominant ADE epitopes. Using a similar design methodology, here we present mRNA-LNP prM-E vaccines for DENV 2, 3, and 4. These vaccines maintained the {Delta}FL modifications, but encode a chimeric E protein to improve VLP expression and stability. All three vaccines elicited neutralizing titers of serotype specific antibodies mice and protected against a lethal homotypic DENV challenge. Mutation of the FL lowered ADE in vaccinated mouse sera. We formulated monotypic vaccines into a tetravalent vaccine and evaluated for immunogenicity and protection in mice. The tetravalent vaccine elicited neutralizing humoral responses against all DENV serotypes and protected against lethal challenges. Cumulatively, our monovalent DENV mRNA-LNP vaccines against all DENV serotypes generate protective immunity and lower the potential for ADE, leading to the development of a first-generation ADE-altered tetravalent DENV vaccine.

microbiology↗

bioRxiv2024

Time-dependent enhancement of mRNA vaccines by 4-1BB costimulation

mRNA vaccines have demonstrated efficacy against COVID-19. However, concerns regarding waning immunity and breakthrough infections have motivated the development of next-generation vaccines with enhanced efficacy. In this study, we investigated the impact of 4-1BB costimulation on immune responses elicited by mRNA vaccines in mice. We first vaccinated mice with an mRNA vaccine encoding the SARS-CoV-2 spike antigen like the Moderna and Pfizer-BioNTech vaccines, followed by administration of 4-1BB costimulatory antibodies at various times post-vaccination. Administering 4-1BB costimulatory antibodies during the priming phase did not enhance immune responses. However, administering 4-1BB costimulatory antibodies after 96 hours elicited a significant improvement in CD8 T cell responses, leading to enhanced protection against breakthrough infections. A similar improvement in immune responses was observed with multiple mRNA vaccines, including vaccines against common cold coronavirus, human immunodeficiency virus (HIV), and arenavirus. These findings demonstrate a time-dependent effect by 4-1BB costimulation and provide insights for developing improved mRNA vaccines.

immunology↗

bioRxiv2023

Caveolin-1 mediates neuroinflammation and cognitive impairment in SARS-CoV-2 infection

Leukocyte infiltration of the CNS can contribute to neuroinflammation and cognitive impairment. Brain endothelial cells regulate adhesion, activation, and diapedesis of T cells across the blood-brain barrier (BBB) in inflammatory diseases. The integral membrane protein Caveolin-1 (Cav-1) critically regulates BBB permeability, but its influence on T cell CNS infiltration in respiratory viral infections is unknown. In this study, we sought to determine the role of Cav-1 at the BBB in neuroinflammation in a COVID-19 mouse model. We used mice genetically deficient in Cav-1 to test the role of this protein in T cell infiltration and cognitive impairment. We found that SARS-CoV-2 infection upregulated brain endothelial Cav-1. Moreover, SARS-CoV-2 infection increased brain endothelial cell vascular cell adhesion molecule-1 (VCAM-1) and CD3+ T cell infiltration of the hippocampus, a region important for short term learning and memory. Concordantly, we observed learning and memory deficits. Importantly, genetic deficiency in Cav-1 attenuated brain endothelial VCAM-1 expression and T cell infiltration in the hippocampus of mice with SARS-CoV-2 infection. Moreover, Cav-1 KO mice were protected from the learning and memory deficits caused by SARS-CoV-2 infection. These results indicate the importance of BBB permeability in COVID-19 neuroinflammation and suggest potential therapeutic value of targeting Cav-1 to improve disease outcomes.

neuroscience↗

bioRxiv2023

Type I IFN stimulates lymph node stromal cells from adult and old mice during a West Nile virus infection

Advanced age is a significant risk factor during viral infection due to an age-associated decline in the immune response. Older individuals are especially susceptible to severe neuroinvasive disease after West Nile virus (WNV) infection. Previous studies have characterized age-associated defects in hematopoietic immune cells during WNV infection that culminate in diminished antiviral immunity. Situated amongst immune cells in the draining lymph node (DLN) are structural networks of nonhematopoietic lymph node stromal cells (LNSCs). LNSCs are comprised of numerous, diverse subsets, with critical roles in the coordination of robust immune responses. The contributions of LNSCs to WNV immunity and immune senescence are unclear. Here, we examine LNSC responses to WNV within adult and old DLNs. Acute WNV infection triggered cellular infiltration and LNSC expansion in adult. Comparatively, aged DLNs exhibited diminished leukocyte accumulation, delayed LNSC expansion, and altered fibroblast and endothelial cell subset composition, signified by fewer LECs. We established an ex vivo culture system to probe LNSC function. Adult and old LNSCs both recognized an ongoing viral infection primarily through type I IFN signaling. Gene expression signatures were similar between adult and old LNSCs. Aged LNSCs were found to constitutively upregulate immediate early response genes. Collectively, these data suggest LNSCs uniquely respond to WNV infection. We are the first to report age-associated differences in LNSCs on the population- and gene expression-level during WNV infection. These changes may compromise antiviral immunity, leading to increased WNV disease in older individuals.

immunology↗

bioRxiv2022

Pre-existing immunity modulates responses to mRNA boosters

mRNA vaccines have shown high efficacy in preventing severe COVID-19, but breakthrough infections, emerging variants and waning antibody levels have warranted the use of boosters. Although mRNA boosters have been widely implemented, the extent to which pre-existing immunity influences the efficacy of boosters remains unclear. In a cohort of individuals primed with the mRNA-1273 or BNT162b2 vaccines, we observed that lower antibody levels before boost were associated with higher fold-increase in antibody levels after boost, suggesting that pre-existing antibody modulates the boosting capacity of mRNA vaccines. Mechanistic studies in mice show that pre-existing antibodies significantly limit antigen expression and priming of B cell responses after mRNA vaccination. Furthermore, we demonstrate that the relative superiority of an updated Omicron vaccine over the original vaccine is critically dependent on the serostatus of the host. These data demonstrate that pre-existing immunity dictates responses to mRNA vaccination, elucidating specific circumstances when updated SARS-CoV-2 vaccines confer superior protection to original vaccines.

immunology↗

bioRxiv2022

Age exacerbates SARS-CoV-2-induced blood-brain barrier leakage and neuropsychiatric dysfunction

Respiratory infection with SARS-CoV-2 causes systemic vascular inflammation and cognitive impairment. We sought to identify the underlying mechanisms mediating vascular dysfunction and inflammation following mild respiratory SARS-CoV-2 infection. To this end, we conduced unbiased transcriptional analysis to identify brain endothelial cell signaling pathways dysregulated by SARS-CoV-2 in vivo. This analysis revealed significant suppression of Wnt/{beta}-catenin signaling, a critical regulator of blood brain barrier integrity. We therefore hypothesized that enhancing cerebrovascular Wnt/{beta}-catenin activity would offer protection against BBB permeability, neuroinflammation, and neurological signs in acute infection. Indeed, we found that delivery of cerebrovascular-targeted, engineered Wnt7a ligands protected blood brain barrier integrity, reduced T cell infiltration of the brain, and reduced microglial activation in SARS-CoV-2 infection. Importantly, this therapeutic strategy also mitigated SARS-CoV-2 induced deficits in the novel object recognition assay for learning and memory and the pole descent task for bradykinesia. These observations suggest that enhancement of Wnt/{beta}-catenin signaling or its downstream effectors could be potential interventional strategies for restoring cognitive health following acute viral infections.

neuroscience↗

bioRxiv2022

Nucleocapsid-specific humoral responses improve the control of SARS-CoV-2

The spike protein of SARS-CoV-2 is a critical antigen present in all approved SARS-CoV-2 vaccines. This surface viral protein is also the target for all monoclonal antibody therapies, but it is unclear whether antibodies targeting other viral proteins can also improve protection against COVID-19. Here, we interrogate whether nucleocapsid-specific antibodies can improve protection against SARS-CoV-2. We first immunized mice with a nucleocapsid-based vaccine, and then transferred sera from these mice into naive mice. On the next day, the recipient mice were challenged intranasally with SARS-CoV-2 to evaluate whether nucleocapsid-specific humoral responses affect viral control. Interestingly, mice that received nucleocapsid-specific sera exhibited enhanced control of a SARS-CoV-2 infection. These findings provide the first demonstration that humoral responses specific to an internal coronavirus protein can help clear infection, warranting the inclusion of other viral antigens in next-generation SARS-CoV-2 vaccines and providing a rationale for the clinical evaluation of nucleocapsid-specific monoclonals to treat COVID-19. HighlightsA SARS-CoV-2 nucleocapsid vaccine elicits robust nucleocapsid-specific antibody responses. This nucleocapsid vaccine generates memory B cells (MBC). Nucleocapsid-specific humoral responses do not prevent SARS-CoV-2 infection. Nucleocapsid-specific humoral responses help control a SARS-CoV-2 infection.

immunology↗

bioRxiv2022

Dual Inhibition of Cathepsin L and 3CL-Pro by GC-376 Constrains SARS Cov2 Infection Including Omicron Variant

Recurrent waves of SARS CoV2 infections remain a major global health concern. Emergence of highly infectious variants with reduced sensitivity to neutralization by vaccines and monoclonal antibodies (mAb) necessitates a deeper understanding of factors involved in SARS CoV2 infections and identification of drug candidates to halt infection. Here, we determined the primacy of endosomal protease cathepsin-L in mediating SARS CoV2 entry and screened a library of well-annotated bioactive compounds for potent cathepsin-L inhibitory activity. Whilst the potent cathepsin-L inhibitors were capable of inhibiting SARS CoV2 entry and cytopathic effect (CPE) in less susceptible cell lines such as human ACE2 expressing 293T cells, these drugs failed to inhibit SARS CoV2 in highly susceptible cell lines such as human TMPRSS2 or human-ACE2-TMPRSS2 overexpressing Vero E6 cells. Only drugs with dual inhibitory effect on both host cathepsin-L and virus 3CL-Protease enzymes such as Z-FA-FMK and GC-376 were capable of inhibiting prototypic (USA-WA1/2020, Lineage A) SARS CoV2 induced CPE in highly susceptible cell lines. Moreover, these drugs inhibited delta (Lineage-B.1.617.2) and omicron (Lineage-B.1.1.529) infection with equal potency showing that the newer mutations harbored in these variants did not affect the mechanism of action of these drugs such as cathepsin-L or 3CL-Pro inhibition. Moreover, our early evidence that 3CL-Pro inhibition can effectively inhibit omicron-induced CPE in highly susceptible cell lines suggests that the recently FDA-approved oral drug, a 3CL-Pro inhibitor which is a combination of nirmatrelvir/ritonavir (Paxlovid) could be effective against omicron variant which shows reduced sensitivity to vaccines and mAb. ImportanceWe report that cathepsin-L and 3CL-Pro as major targets for designing antivirals against SARS CoV2. Dual inhibition of cathepsin-L and 3CL-Pro by GC-376 renders it effective in inhibiting SARS CoV2-induced cytopathic effect in highly susceptible cell lines. Moreover, this candidate drug is equally effective against prototypic SARS CoV2 lineage A and emerging variants such as delta and omicron which show reduced sensitivity to vaccines and monoclonal antibodies. Given the recent wave of SARS CoV2 omicron variant infection around the world, and 3CL-Pro inhibitor nirmatrelvir is one of the components of the FDA-approved Paxlovid, our findings are timely, important and should be of broad interest.

microbiology↗

bioRxiv2021

A mRNA-LNP vaccine against Dengue Virus elicits robust, serotype-specific immunity

Dengue virus (DENV) is the most common vector-borne viral disease with nearly 400 million worldwide infections each year concentrated in the tropical and subtropical regions of the world. Severe dengue complications are often associated with a secondary heterotypic infection of one of the four circulating serotypes. In this scenario, humoral immune responses targeting cross-reactive, poorly-neutralizing epitopes can lead to increased infectivity of susceptible cells via antibody-dependent enhancement (ADE). In this way, antibodies produced in response to infection or vaccination are capable of contributing to enhanced disease in subsequent infections. Currently, there are no available therapeutics to combat DENV disease, and there is an urgent need for a safe and efficacious vaccine. Here, we developed a nucleotide-modified mRNA vaccine encoding for the membrane and envelope structural proteins from DENV serotype 1 encapsulated into lipid nanoparticles (prM/E mRNA-LNP). Vaccination of mice elicited robust antiviral immune responses comparable to viral infection with high levels of neutralizing antibody titers and antiviral CD4+ and CD8+ T cells. Immunocompromised AG129 mice vaccinated with the prM/E mRNA-LNP vaccine were protected from a lethal DENV challenge. Vaccination with either a wild-type vaccine, or a vaccine with mutations in the immunodominant fusion-loop epitope, elicited equivalent humoral and cell mediated immune responses. Neutralizing antibodies elicited by the vaccine were sufficient to protect against a lethal challenge. Both vaccine constructs demonstrated serotype specific immunity with minimal serum cross-reactivity and reduced ADE compared to a live DENV1 viral infection. IMPORTANCEWith 400 million worldwide infections each year, dengue is the most common vector-born viral disease. 40% of the worlds population is at risk with dengue experiencing consistent geographic spread over the years. With no therapeutics available and vaccines performing sub optimally, the need for an effective dengue vaccine is urgent. Here we develop and characterize a novel mRNA vaccine encoding for the dengue serotype 1 envelope and premembrane structural proteins that is delivered via a lipid nanoparticle. Our DENV1 prM/E mRNA-LNP vaccine induces neutralizing antibody and cellular immune responses in immunocompetent mice and protects an immunocompromised mouse from a lethal DENV challenge. Existing antibodies against dengue can enhance subsequent infections via antibody-dependent enhancement. Importantly our vaccine only induced serotype specific immune responses and did not induce ADE.

microbiology↗

bioRxiv2020

mRNA induced expression of human angiotensin-converting enzyme 2 in mice for the study of the adaptive immune response to severe acute respiratory syndrome coronavirus 2

The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic resulting in nearly 20 million infections across the globe, as of August 2020. Critical to the rapid evaluation of vaccines and antivirals is the development of tractable animal models of infection. The use of common laboratory strains of mice to this end is hindered by significant divergence of the angiotensin-converting enzyme 2 (ACE2), which is the receptor required for entry of SARS-CoV-2. In the current study, we designed and utilized an mRNA-based transfection system to induce expression of the hACE2 receptor in order to confer entry of SARS-CoV-2 in otherwise non-permissive cells. By employing this expression system in an in vivo setting, we were able to interrogate the adaptive immune response to SARS-CoV-2 in type 1 interferon receptor deficient mice. In doing so, we showed that the T cell response to SARS-CoV-2 is enhanced when hACE2 is expressed during infection. Moreover, we demonstrated that these responses are preserved in memory and are boosted upon secondary infection. Interestingly, we did not observe an enhancement of SARS-CoV-2 specific antibody responses with hACE2 induction. Importantly, using this system, we functionally identified the CD4+ and CD8+ peptide epitopes targeted during SARS-CoV-2 infection in H2b restricted mice. Antigen-specific CD8+ T cells in mice of this MHC haplotype primarily target peptides of the spike and membrane proteins, while the antigen-specific CD4+ T cells target peptides of the nucleocapsid, membrane, and spike proteins. The functional identification of these T cell epitopes will be critical for evaluation of vaccine efficacy in murine models of SARS-CoV-2. The use of this tractable expression system has the potential to be used in other instances of emerging infections in which the rapid development of an animal model is hindered by a lack of host susceptibility factors.

immunology↗