Biology subjects

Urbanowicz, R. A.

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

7 recordsLinked to original sources

bioRxiv2025

Pre-Omicron Immunity Generates IgG⁺ but Not IgA⁺ Memory B Cells Reactive to Omicron Spike Protein

The emergence of the Omicron variant marked a major antigenic shift from previous SARS-CoV-2 variants. While the emergence of new variants of concern was associated with loss of protection from infection, protection from serious disease was maintained. What was less clear was whether this protection was associated with expansion, affinity maturation of pre-existing cross-reactive B cells, or de novo generation of variant-specific B cells. To define the cross-reactivity of memory B cell populations generated through infection and/or vaccination with pre-Omicron variant spike, we utilised archived samples from healthcare workers enrolled in the extensively monitored Panther study cohort. Using samples collected from Omicron-naive individuals approximately three months (106 days) after a third BNT162b2 vaccine dose, we analyzed the spike-specific B cell repertoire via multiparameter flow cytometry and B cell receptor (BCR) sequencing. Reactivity to both Lineage A and Omicron spike proteins was higher for serum IgG than IgA, and serum neutralisation potency was significantly reduced against pseudoviruses bearing the Omicron spike compared to those with Lineage A spike. The frequency of Omicron-reactive IgA+ B cells was markedly lower than that of Lineage A-reactive cells, whereas Omicron-reactive IgG+ memory B cells were more frequent. These responses were predominantly localized to classical memory and double-negative (DN) B cell subsets. BCR sequencing of donors unexposed to Omicron confirmed that Omicron-binding clones were class-switched and somatically mutated, indicating they originated from pre-existing memory B cells primed by vaccination or infection. Our findings demonstrate that memory B cells generated through infection and/or immunisation with pre-Omicron variants were primed for broad antigenic recognition despite Omicrons extensive genetic divergence. However, the diminished IgA+ B cell frequency suggests a weakened mucosal antibody defence, potentially explaining the continued susceptibility to infection despite preserved protection from severe disease.

immunology↗

bioRxiv2025

An ultra-long heavy chain bovine antibody neutralizes SARS-CoV-2 and reacts broadly with sarbecoviruses.

The ongoing threat of new SARS-CoV-2 variants and other sarbecoviruses has driven efforts to develop broadly neutralizing monoclonal antibodies (mAbs). This study used immunized cattle, known for producing antibodies with ultra-long CDRH3 domains, to generate 33 mAbs, ten of which had ultra-long CDRH3s (>50 amino acids). Of these, mAbs P7 and 99, demonstrated broad and potent neutralization. Notably, mAb P7 neutralized all tested variants, including SARS-CoV-1, with IC50 values between 0.05 and 9.2 {micro}g/mL, and showed cross-reactivity with RBDs from various sarbecoviruses. Structural analysis revealed that mAb 99 binds the spike proteins RBD at the ACE2 binding site. Although the exact binding of P7 wasnt resolved, evidence suggests it targets a hidden epitope, promoting spike trimer dissociation via its extended CDRH3. In Syrian hamsters, both mAbs significantly reduced lung viral loads. These results support the potential of bovine-derived mAbs, particularly those with ultra-long CDRH3s, for future antiviral therapies.

immunology↗

bioRxiv2024

Wisteria floribunda agglutinin enhances Zaire ebolavirus entry through interactions at specific N-linked glycosylation sites on the virus glycoprotein complex

Entry of Zaire ebolavirus (EBOV) into a host cell is a complex process requiring interactions between the viral glycoproteins (GP) and cellular factors. These entry factors are cell-specific and can include cell surface lectins and phosphatidylserine receptors. NPC1 is critical to the late stage of the entry process. Entry has been demonstrated to be enhanced by interactions between the virion and surface-expressed lectins, which interact with carbohydrate moieties attached to the GP. In addition, soluble lectins, including mannose binding lectin (MBL), can enhance entry in vitro. However, the mechanism of lectin-mediated enhancement remains to be defined. This study investigated the potential of three plant lectins, Wisteria floribunda agglutinin (WFA), soybean agglutinin (SBA) and Galanthus nivalis agglutinin (GNA), which possess different carbohydrate binding specificities, to enhance EBOV entry by binding to the GP. WFA was observed to potently enhance entry of lentiviral pseudotype viruses (PVs) expressing the GP of three Ebolavirus species (Zaire, Sudan [SUDV] and Reston [RESTV]), with the greatest impact on EBOV. SBA had a modest enhancing effect on entry that was specific to EBOV, while GNA had no impact on entry of any of the Ebolavirus species. None of the lectins enhanced entry of control PVs expressing the surface proteins of other RNA viruses tested. WFA was demonstrated to bind directly with the EBOV-GP via the glycans, and mutational analysis implicated N238 as contributing to the interaction. Furthermore, enhancement was observed in both human and bat cell lines indicating a highly conserved mechanism of action. We conclude that binding of WFA to EBOV GP through interactions including the glycan at N238 results in GP alterations that enhance entry, providing evidence of a mechanism for lectin-mediated virus entry enhancement. Targeting lectin-ligand interactions presents a potential strategy for restricting Ebolavirus entry.

microbiology↗

bioRxiv2024

Phlorotannin rich Ascophyllum nodosum seaweed extract inhibits influenza infection

Seaweed derived compounds are a renewable resource utilised in the manufacturing and food industry. This study focuses on an Enriched seaweed extract (ESE) isolated from Ascophyllum nodosum. ESE was screened for antiviral activity by plaque reduction assays against influenza A viruses (IAV) H1N1 and H3N2 subtypes. Time of addition assays and FACS analysis were used to help determine the modes of action. The therapeutic potential of ESE was then explored using differentiated human bronchiole epithelial cells at the air liquid interphase and a murine model challenged with IAV. The data indicates ESE primarily interacts directly with virions, preventing virus cell binding. Interestingly, ESE also inhibits early and late stage of the influenza A lifecycle when treatment occurs after cell binding. This inhibitory effect appears to prevent internalisation of virus and release of progeny virus by targeting neuraminidase activity. Intranasal administration of ESE in mice infected with IAV reduced viral load in lung tissue. ESE may be a promising broad acting antiviral agent in the treatment of influenza infections.

microbiology↗

bioRxiv2021

A novel bivalent DNA vaccine encoding both spike protein receptor-binding domain and nucleocapsid protein of SARS-CoV-2 to elicit T cell and neutralising antibody responses that cross react with variants

Although the efficacy of vaccines targeting SARS-CoV-2 is apparent now that the approved mRNA and adenovirus vector vaccines are in widespread use, the longevity of the protective immune response and its efficacy against emerging variants remains to be determined. We have therefore designed a DNA vaccine encoding both the SARS-CoV-2 spike receptorbinding domain ( RBD) and nucleocapsid proteins, the latter of which is highly conserved amongst beta coronaviruses. The vaccine elicits strong pro-inflammatory CD4+ Th1 and CD8+ T-cell responses to both proteins in mice and rats, with responses being significantly enhanced by fusing the nucleocapsid sequence to a modified Fc domain. We have shown that the vaccine also stimulates high titre antibody responses to RBD in mice that efficiently neutralise in pseudotype and live virus neutralisation assays and show cross reactivity with spike proteins from the variants B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). The vaccine also showed good protection in a viral challenge model in ACE2 receptor transgenic mice. This DNA platform can be easily adapted to target variant proteins and we show that a vaccine variant encoding the Beta variant sequence stimulates cross-reactive humoral and T cell responses. These data support the translation of this DNA vaccine platform into the clinic, thereby offering a particular advantage for rapidly targeting emerging SARS-CoV-2 variants.

immunology↗

bioRxiv2020

Immunogenicity of a new gorilla adenovirus vaccine candidate for COVID-19

The COVID-19 pandemic caused by the emergent SARS-CoV-2 coronavirus threatens global public health and there is an urgent need to develop safe and effective vaccines. Here we report the generation and the preclinical evaluation of a novel replication-defective gorilla adenovirus-vectored vaccine encoding the pre-fusion stabilized Spike (S) protein of SARS-CoV2. We show that our vaccine candidate, GRAd- COV2, is highly immunogenic both in mice and macaques, eliciting both functional antibodies which neutralize SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and a robust, Th1- dominated cellular response in the periphery and in the lung. We show here that the pre-fusion stabilized Spike antigen is superior to the wild type in inducing ACE2-interfering, SARS-CoV2 neutralizing antibodies. To face the unprecedented need for vaccine manufacturing at massive scale, different GRAd genome deletions were compared to select the vector backbone showing the highest productivity in stirred tank bioreactors. This preliminary dataset identified GRAd-COV2 as a potential COVID-19 vaccine candidate, supporting the translation of GRAd-COV2 vaccine in a currently ongoing Phase I clinical trial (NCT04528641).

immunology↗

bioRxiv2020

Structure-based design of hepatitis C virus E2 glycoprotein improves serum binding and cross-neutralization

An effective vaccine for hepatitis C virus (HCV) is a major unmet need, and it requires an antigen that elicits immune responses to key conserved epitopes. Based on structures of antibodies targeting HCV envelope glycoprotein E2, we designed immunogens to modulate the structure and dynamics of E2 and favor induction of bNAbs in the context of a vaccine. These designs include a point mutation in a key conserved antigenic site to stabilize its conformation, as well as redesigns of an immunogenic region to add a new N-glycosylation site and mask it from antibody binding. Designs were experimentally characterized for binding to a panel of human monoclonal antibodies (HMAbs) and the coreceptor CD81 to confirm preservation of epitope structure and preferred antigenicity profile. Selected E2 designs were tested for immunogenicity in mice, with and without hypervariable region 1, which is an immunogenic region associated with viral escape. One of these designs showed improvement in polyclonal immune serum binding to HCV pseudoparticles and neutralization of isolates associated with antibody resistance. These results indicate that antigen optimization through structure-based design of the envelope glycoproteins is a promising route to an effective vaccine for HCV.

microbiology↗