Biology subjects

Lee, W. S.

Publications and source records attributed to Lee, W. S..

11 recordsLinked to original sources

bioRxiv2026

Immunogen selection and prior immunity shape antibody breadth following immunisation with avian H5 hemagglutinin

Avian influenza A viruses pose a persistent zoonotic threat to humans owing to their expanding host range and high case fatality rates. In particular, viruses from the 2.3.4.4b clade of the H5 subtype have now been detected in over 60 mammalian species, raising serious pandemic concerns. Understanding immune recognition of the H5 hemagglutinin (HA) is therefore critical for effective vaccine design and pandemic preparedness. To understand the breadth of cross-recognition induced by different H5 strains, we selected genetically diverse H5 human isolates from 2003-2023 and assessed neutralising antibody responses elicited by adjuvanted recombinant HA protein-based vaccines in C57BL/6 mice. Neutralisation activity of sera was determined against seven H5 HA variants using pseudotyped viruses and a PR8-reassortant virus in micro-neutralisation assays. Our results showed a wide variety of cross-strain neutralisation across H5 HA antigen variants. The conventional vaccine strain A/Indonesia/05/2005 displayed narrow activity against emerging clade 2.3.4.4b viruses, whereas ancestral variants exhibited cross-neutralisation profiles showing a diversity of breath but with limited potency. Polyvalent H5 HA formulations and nanoparticle-displayed H5 HA platforms substantially broadened cross-neutralisation against diverse H5 strains. To examine the impact of pre-existing immunity on H5 vaccine immunogenicity in mouse models, mice were primed with either seasonal influenza infection or quadrivalent influenza vaccine (QIV) prior to H5 HA immunisation. QIV pre-vaccination, but not prior influenza infection, enhanced subsequent neutralizing responses towards A/Fujian-Sanyuan/21099/2017 (clade 2.3.4.4b) H5. Collectively, our results demonstrate that immunogen selection and prior immunity shape antibody breadth following immunisation with avian A(H5) hemagglutinin.

immunology↗

bioRxiv2025

De novo administration of antiviral monoclonal antibodies against SARS-CoV-2 or influenza using mRNA lipid nanoparticles

Monoclonal antibodies (mAbs) are an emerging class of therapeutics for the prevention and treatment of viral infections. Recent advances in mRNA/lipid nanoparticle (LNP) technology provide a potential new modality for the expression of mAbs in vivo, potentially bypassing the need for recombinant manufacturing of mAb proteins. In this study, we compared traditional infusion of neutralising mAbs targeting SARS-CoV-2 or influenza to mRNA-based induction of de novo mAb expression in treated mice. High serum concentrations of mAbs were achieved upon delivery of a single mRNA encoding both heavy and light chains via intravenous or intramuscular routes using prototypic LNP formulations. However, pharmacokinetics were heavily influenced by the induction of anti-drug antibody responses directed against the encoded mAbs, driving reductions in in vivo half-life and compromising protective capacity against SARS-CoV-2 Omicron BA.1 infection. Overall, mRNA/LNP delivery comprises a feasible and attractive pathway to speed the development and deployment of antiviral antibodies, however optimisation of LNP formulation, dosing and administration routes is required to maximise protective potential.

immunology↗

bioRxiv2025

A novel chimeric coronavirus spike vaccine combining SARS-CoV-2 RBD and scaffold domains from HKU-1 elicits potent neutralising antibody responses

The receptor binding domain (RBD) of the SARS-CoV-2 spike is the major target for neutralising antibodies elicited by current vaccines. Using small domains such as the RBD as vaccine immunogens, however, may constrain the availability of CD4 T follicular helper (TFH) cells and impact immunogenicity. We engineered a novel chimeric trimeric RBD (CTR) glycoprotein, replacing the RBD of human coronavirus HKU-1 spike with SARS-CoV-2 RBD of either ancestral (WT) or Omicron BA.2 strains. This strategy maintains a native trimeric conformation of the RBD, while providing additional sources of CD4 T cell help via the HKU-1 spike scaffold. In C57BL/6 mice, CTR-BA.2 prime-boost vaccination elicited high anti-BA.2-RBD IgG and neutralising titres, matching responses in animals immunised with native SARS-CoV-2 spike proteins. GC B cells elicited by CTR-BA.2 were predominantly WT+/BA.2+ cross- reactive, and TFH cells predominantly recognised HKU-1 epitopes, demonstrating scaffold-directed T cell help. Macaques prime-boost immunised with CTR-WT similarly elicited high anti-RBD IgG, anti-spike IgG and neutralising responses, comparable to native spike-vaccinated animals. In draining lymph nodes of CTR-WT vaccinated macaques, RBD-specific GC B cells were present at elevated levels. In contrast to the murine studies, lymph node-draining TFH responses in macaques were broadly elicited against RBD, NTD/S2 or HKU-1-derived peptides. Although native SARS- CoV-2 spike was also highly immunogenic in animal models, our findings establish the chimeric glycoprotein design as a strategy to overcome the poor immunogenicity of the SARS-CoV-2 RBD by engaging CD4 TFH cells, while maintaining the ability to elicit protective neutralising responses. One sentence summaryA chimeric glycoprotein design preserves SARS-CoV-2 RBD antigenic conformation enabling elicitation of neutralising responses, while allowing recruitment of HKU-1 scaffold-directed CD4 helper responses to support the humoral response.

immunology↗

bioRxiv2025

Contemporary seasonal human coronaviruses display differences in cellular tropism compared to laboratory-adapted reference strains

Seasonal human coronaviruses (sHCoVs) cause 15-30% of common colds. The reference strains used for research were isolated decades ago and have been passaged extensively but contemporary sHCoVs have been challenging to study as they are notoriously difficult to grow in standard immortalized cell lines. Here we addressed these issues by utilizing primary human nasal epithelial cells (HNECs) and immortalized human bronchial epithelial cells (BCi) differentiated at an air-liquid interface, as well as human embryonic stem cell-derived alveolar type II (AT2) cells to recover contemporary sHCoVs from human nasopharyngeal specimens. From 21 specimens we recovered four 229E, three NL63 and eight OC43 viruses. All contemporary sHCoVs showed sequence differences from lab-adapted CoVs, particularly within the spike gene. Evidence of nucleotide changes in the receptor binding domains within 229E and detection of recombination for both 229E and OC43 isolates was also observed. Importantly, we developed methods for the amplification of high titre stocks of NL63 and 229E, that maintained sequence identity, and we established methods for the titration of contemporary sHCoV isolates. Comparison of lab-adapted and contemporary strains in immortalised cell lines and airway epithelial cells revealed differences in cell tropism, growth kinetics and cytokine production between lab-adapted and contemporary sHCoV strains. These data confirm that contemporary sHCoVs differ from lab-adapted reference strains and, using the methods established here, should be used for study of CoV biology and evaluation of medical countermeasures. ImportanceZoonotic coronaviruses have caused significant public health emergencies. The occurrence of a similar spillover event in the future is likely and efforts to further understand coronavirus biology should be a high priority. Several seasonal coronaviruses circulate within the human population. Efforts to study these viruses have been limited to reference strains isolated decades ago due to the difficulty in isolating clinical isolates. Here, we use human airway and alveolar epithelial cultures to recover contemporary isolates of NL63, 229E and OC43. We establish methods to make high titre stocks and titrate 229E and NL63 isolates. We show that contemporary isolates of NL63 and OC43 have a different tropism within the respiratory epithelium compared to lab-adapted strains. Although 229E clinical and lab-adapted strains similarly infect the respiratory epithelium, differences in host response and replication kinetics are observed. Using the methods developed here, future research should include contemporary isolates when studying coronavirus biology.

microbiology↗

bioRxiv2023

Monoclonal antibodies against human coronavirus NL63 spike

The COVID-19 pandemic has illustrated the potential for monoclonal antibody therapeutics as prophylactic and therapeutic agents against pandemic viruses. No such therapeutics currently exist for other human coronaviruses. NL63 is a human alphacoronavirus that typically causes the common cold and uses the same receptor, ACE2, as the highly pathogenic SARS-CoV and SARS-CoV-2 pandemic viruses. In a cohort of healthy adults, we characterised humoral responses against the NL63 spike protein. While NL63 spike and receptor binding domain-specific binding antibodies and neutralisation activity could be detected in plasma of all subjects, memory B cells against NL63 spike were variable and relatively low in frequency compared to that against SARS-CoV-2 spike. From these donors, we isolated a panel of antibodies against NL63 spike and characterised their neutralising potential. We identified potent neutralising antibodies that recognised the receptor binding domain (RBD) and other non-RBD epitopes within spike.

microbiology↗

bioRxiv2022

Rapid recall and de novo T cell responses during SARS-CoV-2 breakthrough infection

While the protective role of neutralising antibodies against COVID-19 is well-established, questions remain about the relative importance of cellular immunity. Using 6 pMHC-multimers in a cohort with early and frequent sampling we define the phenotype and kinetics of recalled and primary T cell responses following Delta or Omicron breakthrough infection. Recall of spike-specific CD4+ T cells was rapid, with cellular proliferation and extensive activation evident as early as 1 day post-symptom onset. Similarly, spike-specific CD8+ T cells were rapidly activated but showed variable levels of expansion. Strikingly, high levels of SARS-CoV-2-specific CD8+ T cell activation at baseline and peak were strongly correlated with reduced peak SARS-CoV-2 RNA levels in nasal swabs and accelerated clearance of virus. Our study demonstrates rapid and extensive recall of memory T cell populations occurs early after breakthrough infection and suggests that CD8+ T cells contribute to the control of viral replication in breakthrough SARS-CoV-2 infections.

immunology↗

bioRxiv2020

Prime-boost protein subunit vaccines against SARS-CoV-2 are highly immunogenic in mice and macaques

SARS-CoV-2 vaccines are advancing into human clinical trials, with emphasis on eliciting high titres of neutralising antibodies against the viral spike (S). However, the merits of broadly targeting S versus focusing antibody onto the smaller receptor binding domain (RBD) are unclear. Here we assessed prototypic S and RBD subunit vaccines in homologous or heterologous prime-boost regimens in mice and non-human primates. We find S is highly immunogenic in mice, while the comparatively poor immunogenicity of RBD was associated with limiting germinal centre and T follicular helper cell activity. Boosting S-primed mice with either S or RBD significantly augmented neutralising titres, with RBD-focussing driving moderate improvement in serum neutralisation. In contrast, both S and RBD vaccines were comparably immunogenic in macaques, eliciting serological neutralising activity that generally exceed levels in convalescent humans. These studies confirm recombinant S proteins as promising vaccine candidates and highlight multiple pathways to achieving potent serological neutralisation.

immunology↗

bioRxiv2020

Coformulation with tattoo ink for immunological assessment of vaccine immunogenicity in the draining lymph node

Characterisation of germinal centre B and T cell responses yields critical insights into vaccine immunogenicity. Non-human primates are a key pre-clinical animal model for human vaccine development, allowing both lymph node and circulating immune responses to be longitudinally sampled for correlates of vaccine efficacy. However, patterns of vaccine antigen drainage via the lymphatics after intramuscular immunisation can be stochastic, driving uneven deposition between lymphoid sites, and between individual lymph nodes within larger clusters. In order to improve the accurate isolation of antigen-exposed lymph nodes during biopsies and necropsies, we developed and validated a method for co-formulating candidate vaccines with tattoo ink, which allows for direct visual identification of vaccine-draining lymph nodes and evaluation of relevant antigen-specific B and T cell responses by flow cytometry. This approach improves the assessment of vaccine-induced immunity in highly relevant non-human primate models.

immunology↗

bioRxiv2020

Is FAM19A5 an adipokine? Peripheral FAM19A5 in wild-type, FAM19A5 knock-out, and LacZ knock-in mice.

FAM19A5 is a novel secretory protein primarily expressed in the brain. However, a recent study reported that FAM19A5 is an adipocyte-derived adipokine that regulates vascular smooth muscle function through sphingosine-1-phosphate receptor 2 (S1PR2). In our study, we investigated FAM19A5 transcript and protein levels in peripheral tissues, including adipose tissues, from wild-type, FAM19A5 knockout, and FAM19A5 LacZ knockin mice. We found that FAM19A5 transcript levels in the central nervous system were much greater than those in any of the peripheral tissues, including adipose tissues. Furthermore, the FAM19A5 protein levels in adipose and reproductive tissues were below detectable limits for Western blot analysis. Additionally, we found that the FAM19A5 protein did not interact with S1PR2 in terms of G protein-mediated signal transduction, {beta}-arrestin recruitment, or ligand-mediated internalization. Taken together, our findings revealed basal levels of FAM19A5 transcripts and proteins in peripheral tissues, confirming its primary expression in the central nervous system and lack of significant interaction with S1PR2.

genetics↗

bioRxiv2019

Dysmorphic neuron density is highest in the centre of epileptogenic cortical tubers

Cortical tubers are benign lesions that develop in patients with tuberous sclerosis complex (TSC), often resulting in drug-resistant epilepsy. Surgical resection may be required for seizure control, but the extent of the resection required is unclear. Many centres include resection of perituberal cortex, which may be associated with neurological deficits. Also, patients with tubers in eloquent cortex may be excluded from epilepsy surgery.\n\nOur electrophysiological and MRI studies indicate that the tuber centre is the source of seizures, suggesting that smaller resections may be sufficient for seizure control. Here we report five epilepsy surgeries in four children with TSC and focal motor seizures from solitary epileptogenic tubers in the sensorimotor cortex in whom the resection was limited to the tuber centre, leaving the tuber rim and surrounding perituberal cortex intact. Seizures were eliminated in all cases, and no functional deficits were observed. On routine histopathology we observed an apparent increase in density of dysmorphic neurons at the tuber centre, which we confirmed using unbiased stereology which demonstrated a significantly greater density of dysmorphic neurons within the resected tuber centre (1951 {+/-} 215 cells/mm3) compared to the biopsied tuber rim (531 {+/-} 189 cells/mm3, n = 4, p = 0.008).\n\nTaken together with our previous electrophysiological and MRI studies implicating the tuber centre as the focus of epileptic activity, and other electrophysiological studies of dysmorphic neurons in focal cortical dysplasia, this study supports the hypothesis that dysmorphic neurons concentrated at the tuber centre are the seizure generators in TSC. Furthermore, our results support limiting resection to the tuber centre, decreasing the risk of neurological deficits when tubers are located within eloquent cortex.

neuroscience↗

bioRxiv2016

UNC-5 (UNC5) Regulates the Length and Number of Processes that Caenorhabditis elegans Neurons Can Develop

Neurons extend processes that vary in number, length, and direction of outgrowth. Extracellular cues help determine outgrowth patterns. In Caenorhabditis elegans, neurons respond to the extracellular UNC-6 (netrin) cue via UNC-40 (DCC) and UNC-5 (UNC5) receptors. Previously we presented evidence that UNC-40 asymmetric localization at the plasma membrane is self-organizing and that UNC-40 can localize and mediate outgrowth at randomly selected sites. We also postulate that the process is statistically dependent, i.e. if the probability of outgrowth at one site changes then the probability at another site(s) must also change. Over time, the direction of outgrowth activity fluctuates across the membrane. A probability distribution describes the likelihood of outgrowth in each direction. Random walk modeling predicts that the degree to which the direction of outgrowth fluctuations affects the outward displacement of the membrane. We predict that extracellular cues create patterns of outgrowth by differentially affecting the degree to which the direction of outgrowth activity fluctuates along the membrane. This produces different rates of outgrowth along the surface and creates patterns of extension. Here we present evidence that UNC-5 (UNC5) receptor activity regulates UNC-40 asymmetric localization and the patterning of outgrowth. We show that unc-5 mutations alter UNC-40 asymmetric localization and the patterns of outgrowth that neurons develop. Genetic interactions suggest UNC-5 acts through the UNC-53 (NAV2) cytoplasmic protein to regulate UNC-40 asymmetric localization in response to both the UNC-6 and EGL-20 (wnt) extracellular cues.

neuroscience↗