Equal Maintenance of Anti-SARS-CoV-2 Antibody Levels Induced by Heterologous and Homologous Regimens of the BNT162b2, ChAdOx1, CoronaVac and Ad26.COV2.S Vaccines: A Longitudinal Study Up to the 4th Dose of Booster.

Several technological approaches have been used to develop vaccines against COVID-19, including those based on inactivated viruses, viral vectors, and mRNA. This study aimed to monitor the maintenance of anti-SARS-CoV-2 antibodies in individuals from Brazil according to the primary vaccination regimen, as follows: BNT162b2 (group 1; 22) and ChAdOx1 (group 2; 18). Everyone received BNT162b2 in the first booster while in the second booster CoronaVac, Ad26.COV2.S, or BNT162b2. Blood samples were collected from 2021 to 2023 to analyze specific RBD (ELISA) and neutralizing antibodies (PRNT50). We observed a progressive increase in anti-RBD and neutralizing antibodies in each subsequent dose, remaining at high titers until the end of follow-up. Group 1 had higher anti-RBD antibody titers than group 2 after beginning the primary regimen, with significant differences after the 2nd and 3rd doses. Group 2 showed a more expressive increase after the first booster with BNT162B2 (heterologous booster). Group 2 also presented high levels of neutralizing antibodies against the Gamma and Delta variants until five months after the second booster. In conclusion, the circulating levels of anti-RBD and neutralizing antibodies against the two variants of SARS-CoV-2 were durable even five months after the 4th dose, suggesting that periodic booster vaccinations (homologous or heterologous) induced long-lasting immunity.

A chimeric vaccine derived from Australian genotype IV Japanese encephalitis virus protects mice from lethal challenge.

In 2022, a genotype IV (GIV) strain of Japanese encephalitis virus (JEV) caused an unprecedented and widespread outbreak of disease in pigs and humans in Australia. As no veterinary vaccines against JEV are approved in Australia and all current approved human and veterinary vaccines are derived from genotype (G) III JEV strains, we used the recently described insect-specific Binjari virus (BinJV) chimeric flavivirus vaccine technology to produce a JEV GIV vaccine candidate. Herein we describe the production of a chimeric virus displaying the structural prM and E proteins of a JEV GIV isolate obtained from a stillborn piglet (JEVNSW/22) in the genomic backbone of BinJV (BinJ/JEVNSW/22-prME). BinJ/JEVNSW/22-prME was shown to be antigenically indistinguishable from the JEVNSW/22 parental virus by KD analysis and a panel of JEV-reactive monoclonal antibodies in ELISA. BinJ/JEVNSW/22-prME replicated efficiently in C6/36 cells, reaching titres of >107 infectious units/mL - an important attribute for vaccine manufacture. As expected, BinJ/JEVNSW/22-prME failed to replicate in a variety of vertebrate cells lines. When used to immunise mice, the vaccine induced a potent virus neutralising response against JEVNSW/22 and to GII and GIII JEV strains. The BinJ/JEVNSW/22-prME vaccine provided complete protection against lethal challenge with JEVNSW/22, whilst also providing partial protection against viraemia and disease for the related Murray Valley encephalitis virus. Our results demonstrate that BinJ/JEVNSW/22-prME is a promising vaccine candidate against JEV.

Amphiphilic Heparinoids as Potent Antiviral Agents against SARS-CoV-2.

Herein, we report the synthesis and biological evaluation of a novel series of heparinoid amphiphiles as inhibitors of heparanase and SARS-CoV-2. By employing a tailor-made synthetic strategy, a library of highly sulfated homo-oligosaccharides bearing d-glucose or a C5-epimer (i.e., l-idose or l-iduronic acid) conjugated with various lipophilic groups was synthesized and investigated for antiviral activity. Sulfated higher oligosaccharides of d-glucose or l-idose with lipophilic aglycones displayed potent anti-SARS-CoV-2 and antiheparanse activity, similar to or better than pixatimod (PG545), and were more potent than their isosteric l-iduronic acid congeners. Lipophilic groups such as cholestanol and C18-aliphatic substitution are more advantageous than functional group appended lipophilic moieties. These findings confirm that fine-tuning of higher oligosaccharides, degree of sulfation, and lipophilic groups can yield compounds with potent anti-SARS-CoV-2 activity.

Antiviral Activities of Heparan Sulfate Mimetic RAFT Polymers Against Mosquito-borne Viruses.

Mosquito-borne viruses are a major worldwide health problem associated with high morbidity and mortality rates and significant impacts on national healthcare budgets. The development of antiviral drugs for both the treatment and prophylaxis of these diseases is thus of considerable importance. To address the need for therapeutics with antiviral activity, a library of heparan sulfate mimetic polymers was screened against dengue virus (DENV), Yellow fever virus (YFV), Zika virus (ZIKV), and Ross River virus (RRV). The polymers were prepared by RAFT polymerization of various acidic monomers with a target MW of 20 kDa (average Mn ∼ 27 kDa by GPC). Among the polymers, poly(SS), a homopolymer of sodium styrenesulfonate, was identified as a broad spectrum antiviral with activity against all the tested viruses and particularly potent inhibition of YFV (IC50 = 310 pM). Our results further uncovered that poly(SS) exhibited a robust inhibition of ZIKV infection in both mosquito and human cell lines, which points out the potential functions of poly(SS) in preventing mosquito-borne viruses associated diseases by blocking viral transmission in their mosquito vectors and mitigating viral infection in patients.

Enhancement of cellular immunity following needle-free vaccination of mice with SARS-CoV-2 spike protein.

The COVID-19 pandemic has highlighted the need for vaccines capable of providing rapid and robust protection. One way to improve vaccine efficacy is delivery via microarray patches, such as the Vaxxas high-density microarray patch (HD-MAP). We have previously demonstrated that delivery of a SARS-CoV-2 protein vaccine candidate, HexaPro, via the HD-MAP induces potent humoral immune responses. Here, we investigate the cellular responses induced by HexaPro HD-MAP vaccination. We found that delivery via the HD-MAP induces a type one biassed cellular response of much greater magnitude as compared to standard intramuscular immunization.

The role of N-glycosylation in spike antigenicity for the SARS-CoV-2 gamma variant.

The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity towards the viral spike protein, whether acquired from infection or vaccination. Mutations that impact N-glycosylation of spike may be particularly important in influencing antigenicity, but their consequences are difficult to predict. Here, we compare the glycosylation profiles and antigenicity of recombinant viral spike of ancestral Wu-1 and the Gamma strain, which has two additional N-glycosylation sites due to amino acid substitutions in the N-terminal domain (NTD). We found that a mutation at residue 20 from threonine to asparagine within the NTD caused the loss of NTD-specific antibody COVA2-17 binding. Glycan site-occupancy analyses revealed that the mutation resulted in N-glycosylation switching to the new sequon at N20 from the native N17 site. Site-specific glycosylation profiles demonstrated distinct glycoform differences between Wu-1, Gamma, and selected NTD variant spike proteins, but these did not affect antibody binding. Finally, we evaluated the specificity of spike proteins against convalescent COVID-19 sera and found reduced cross-reactivity against some mutants, but not Gamma spike compared to Wuhan spike. Our results illustrate the impact of viral divergence on spike glycosylation and SARS-CoV-2 antibody binding profiles.

Senolytic therapy alleviates physiological human brain aging and COVID-19 neuropathology.

Aging is a major risk factor for neurodegenerative diseases, and coronavirus disease 2019 (COVID-19) is linked to severe neurological manifestations. Senescent cells contribute to brain aging, but the impact of virus-induced senescence on neuropathologies is unknown. Here we show that senescent cells accumulate in aged human brain organoids and that senolytics reduce age-related inflammation and rejuvenate transcriptomic aging clocks. In postmortem brains of patients with severe COVID-19 we observed increased senescent cell accumulation compared with age-matched controls. Exposure of human brain organoids to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induced cellular senescence, and transcriptomic analysis revealed a unique SARS-CoV-2 inflammatory signature. Senolytic treatment of infected brain organoids blocked viral replication and prevented senescence in distinct neuronal populations. In human-ACE2-overexpressing mice, senolytics improved COVID-19 clinical outcomes, promoted dopaminergic neuron survival and alleviated viral and proinflammatory gene expression. Collectively our results demonstrate an important role for cellular senescence in driving brain aging and SARS-CoV-2-induced neuropathology, and a therapeutic benefit of senolytic treatments.

Kinetics of severe dengue virus infection and development of gut pathology in mice.

IMPORTANCE: Dengue virus, an arbovirus, causes an estimated 100 million symptomatic infections annually and is an increasing threat as the mosquito range expands with climate change. Dengue epidemics are a substantial strain on local economies and health infrastructure, and an understanding of what drives severe disease may enable treatments to help reduce hospitalizations. Factors exacerbating dengue disease are debated, but gut-related symptoms are much more frequent in severe than mild cases. Using mouse models of dengue infection, we have shown that inflammation and damage are earlier and more severe in the gut than in other tissues. Additionally, we observed impairment of the gut mucus layer and propose that breakdown of the barrier function exacerbates inflammation and promotes severe dengue disease. This idea is supported by recent data from human patients showing elevated bacteria-derived molecules in dengue patient serum. Therapies aiming to maintain gut integrity may help to abrogate severe dengue disease.

Long-term safety and immunogenicity of an MF59-adjuvanted spike glycoprotein-clamp vaccine for SARS-CoV-2 in adults aged 18-55 years or ≥56 years: 12-month results from a randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND: We previously demonstrated the safety and immunogenicity of an MF59-adjuvanted COVID-19 vaccine based on the SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a molecular clamp using HIV-1 glycoprotein 41 sequences. Here, we describe 12-month results in adults aged 18-55 years and ≥56 years. METHODS: Phase 1, double-blind, placebo-controlled trial conducted in Australia (July 2020-December 2021; ClinicalTrials.govNCT04495933; active, not recruiting). Healthy adults (Part 1: 18-55 years; Part 2: ≥56 years) received two doses of placebo, 5 µg, 15 µg, or 45 µg vaccine, or one 45 µg dose of vaccine followed by placebo (Part 1 only), 28 days apart (n = 216; 24 per group). Safety, humoral immunogenicity (including against virus variants), and cellular immunogenicity were assessed to day 394 (12 months after second dose). Effects of subsequent COVID-19 vaccination on humoral responses were examined. FINDINGS: All two-dose vaccine regimens were well tolerated and elicited strong antigen-specific and neutralising humoral responses, and CD4+ T-cell responses, by day 43 in younger and older adults, although cellular responses were lower in older adults. Humoral responses waned by day 209 but were boosted in those receiving authorised vaccines. Neutralising activity against Delta and Omicron variants was present but lower than against the Wuhan strain. Cross-reactivity in HIV diagnostic tests declined over time but remained detectable in most participants. INTERPRETATION: The SARS-CoV-2 molecular clamp vaccine is well tolerated and evokes robust immune responses in adults of all ages. Although the HIV glycoprotein 41-based molecular clamp is not being progressed, the clamp concept represents a viable platform for vaccine development. FUNDING: This study was funded by the Coalition for Epidemic Preparedness Innovations, the National Health and Medical Research Council of Australia, and the Queensland Government.

A nanobody recognizes a unique conserved epitope and potently neutralizes SARS-CoV-2 omicron variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) Omicron variant sub-lineages spread rapidly worldwide, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for effective anti-SARS-CoV-2 agents against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production, and potential for delivery via inhalation. Here, we characterize the receptor binding domain (RBD)-specific nanobody W25 and show superior neutralization activity toward Omicron sub-lineages in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse W25 for further clinical development.

Structure and antigenicity of divergent Henipavirus fusion glycoproteins.

In August 2022, a novel henipavirus (HNV) named Langya virus (LayV) was isolated from patients with severe pneumonic disease in China. This virus is closely related to Mòjiang virus (MojV), and both are divergent from the bat-borne HNV members, Nipah (NiV) and Hendra (HeV) viruses. The spillover of LayV is the first instance of a HNV zoonosis to humans outside of NiV and HeV, highlighting the continuing threat this genus poses to human health. In this work, we determine the prefusion structures of MojV and LayV F proteins via cryogenic electron microscopy to 2.66 and 3.37 Å, respectively. We show that despite sequence divergence from NiV, the F proteins adopt an overall similar structure but are antigenically distinct as they do not react to known antibodies or sera. Glycoproteomic analysis revealed that while LayV F is less glycosylated than NiV F, it contains a glycan that shields a site of vulnerability previously identified for NiV. These findings explain the distinct antigenic profile of LayV and MojV F, despite the extent to which they are otherwise structurally similar to NiV. Our results carry implications for broad-spectrum HNV vaccines and therapeutics, and indicate an antigenic, yet not structural, divergence from prototypical HNVs.

Inactivation of SARS-CoV-2 infectivity in platelet concentrates or plasma following treatment with ultraviolet C light or with methylene blue combined with visible light.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unlikely to be a major transfusion-transmitted pathogen; however, convalescent plasma is a treatment option used in some regions. The risk of transfusion-transmitted infections can be minimized by implementing Pathogen Inactivation (PI), such as THERAFLEX MB-plasma and THERAFLEX UV-Platelets systems. Here we examined the capability of these PI systems to inactivate SARS-CoV-2. STUDY DESIGN AND METHODS: SARS-CoV-2 spiked plasma units were treated using the THERAFLEX MB-Plasma system in the presence of methylene blue (~0.8 µmol/L; visible light doses: 20, 40, 60, and 120 [standard] J/cm2 ). SARS-CoV-2 spiked platelet concentrates (PCs) were treated using the THERAFLEX UV-platelets system (UVC doses: 0.05, 0.10, 0.15, and 0.20 [standard] J/cm2 ). Samples were taken prior to the first and after each illumination dose, and viral infectivity was assessed using an immunoplaque assay. RESULTS: Treatment of spiked plasma with the THERAFLEX MB-Plasma system resulted in an average ≥5.03 log10 reduction in SARS-CoV-2 infectivity at one third (40 J/cm2 ) of the standard visible light dose. For the platelet concentrates (PCs), treatment with the THERAFLEX UV-Platelets system resulted in an average ≥5.18 log10 reduction in SARS-CoV-2 infectivity at the standard UVC dose (0.2 J/cm2 ). CONCLUSIONS: SARS-CoV-2 infectivity was reduced in plasma and platelets following treatment with the THERAFLEX MB-Plasma and THERAFLEX UV-Platelets systems, to the limit of detection, respectively. These PI technologies could therefore be an effective option to reduce the risk of transfusion-transmitted emerging pathogens.

SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein.

Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson's disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation. Using SARS-CoV-2 infection of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) as a COVID-19 pre-clinical model, we established the presence of virus in the brain together with microglial activation and NLRP3 inflammasome upregulation in comparison to uninfected mice. Next, utilising a model of human monocyte-derived microglia, we identified that SARS-CoV-2 isolates can bind and enter human microglia in the absence of viral replication. This interaction of virus and microglia directly induced robust inflammasome activation, even in the absence of another priming signal. Mechanistically, we demonstrated that purified SARS-CoV-2 spike glycoprotein activated the NLRP3 inflammasome in LPS-primed microglia, in a ACE2-dependent manner. Spike protein also could prime the inflammasome in microglia through NF-κB signalling, allowing for activation through either ATP, nigericin or α-synuclein. Notably, SARS-CoV-2 and spike protein-mediated microglial inflammasome activation was significantly enhanced in the presence of α-synuclein fibrils and was entirely ablated by NLRP3-inhibition. Finally, we demonstrate SARS-CoV-2 infected hACE2 mice treated orally post-infection with the NLRP3 inhibitory drug MCC950, have significantly reduced microglial inflammasome activation, and increased survival in comparison with untreated SARS-CoV-2 infected mice. These results support a possible mechanism of microglial innate immune activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson's disease in COVID-19 infected individuals, and a potential therapeutic avenue for intervention.

Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis.

All flaviviruses, including Zika virus, produce noncoding subgenomic flaviviral RNA (sfRNA), which plays an important role in viral pathogenesis. However, the exact mechanism of how sfRNA enables viral evasion of antiviral response is not well defined. Here, we show that sfRNA is required for transplacental virus dissemination in pregnant mice and subsequent fetal brain infection. We also show that sfRNA promotes apoptosis of neural progenitor cells in human brain organoids, leading to their disintegration. In infected human placental cells, sfRNA inhibits multiple antiviral pathways and promotes apoptosis, with signal transducer and activator of transcription 1 (STAT1) identified as a key shared factor. We further show that the production of sfRNA leads to reduced phosphorylation and nuclear translocation of STAT1 via a mechanism that involves sfRNA binding to and stabilizing viral protein NS5. Our results suggest the cooperation between viral noncoding RNA and a viral protein as a novel strategy for counteracting antiviral responses.

Characterization and comparison of novel adjuvants for a prefusion clamped MERS vaccine.

Various chemical adjuvants are available to augment immune responses to non-replicative, subunit vaccines. Optimized adjuvant selection can ensure that vaccine-induced immune responses protect against the diversity of pathogen-associated infection routes, mechanisms of infectious spread, and pathways of immune evasion. In this study, we compare the immune response of mice to a subunit vaccine of Middle Eastern respiratory syndrome coronavirus (MERS-CoV) spike protein, stabilized in its prefusion conformation by a proprietary molecular clamp (MERS SClamp) alone or formulated with one of six adjuvants: either (i) aluminium hydroxide, (ii) SWE, a squalene-in-water emulsion, (iii) SQ, a squalene-in-water emulsion containing QS21 saponin, (iv) SMQ, a squalene-in-water emulsion containing QS21 and a synthetic toll-like receptor 4 (TLR4) agonist 3D-6-acyl Phosphorylated HexaAcyl Disaccharide (3D6AP); (v) LQ, neutral liposomes containing cholesterol, 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and QS21, (vi) or LMQ, neutral liposomes containing cholesterol, DOPC, QS21, and 3D6AP. All adjuvanted formulations induced elevated antibody titers which where greatest for QS21-containing formulations. These had elevated neutralization capacity and induced higher frequencies of IFNƔ and IL-2-producing CD4+ and CD8+ T cells. Additionally, LMQ-containing formulations skewed the antibody response towards IgG2b/c isotypes, allowing for antibody-dependent cellular cytotoxicity. This study highlights the utility of side-by-side adjuvant comparisons in vaccine development.

A platform technology for generating subunit vaccines against diverse viral pathogens.

The COVID-19 pandemic response has shown how vaccine platform technologies can be used to rapidly and effectively counteract a novel emerging infectious disease. The speed of development for mRNA and vector-based vaccines outpaced those of subunit vaccines, however, subunit vaccines can offer advantages in terms of safety and stability. Here we describe a subunit vaccine platform technology, the molecular clamp, in application to four viruses from divergent taxonomic families: Middle Eastern respiratory syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), Lassa virus (LASV) and Nipah virus (NiV). The clamp streamlines subunit antigen production by both stabilising the immunologically important prefusion epitopes of trimeric viral fusion proteins while enabling purification without target-specific reagents by acting as an affinity tag. Conformations for each viral antigen were confirmed by monoclonal antibody binding, size exclusion chromatography and electron microscopy. Notably, all four antigens tested remained stable over four weeks of incubation at 40°C. Of the four vaccines tested, a neutralising immune response was stimulated by clamp stabilised MERS-CoV spike, EBOV glycoprotein and NiV fusion protein. Only the clamp stabilised LASV glycoprotein precursor failed to elicit virus neutralising antibodies. MERS-CoV and EBOV vaccine candidates were both tested in animal models and found to provide protection against viral challenge.

SARS-CoV-2 triggers complement activation through interactions with heparan sulfate.

Objectives: To determine whether SARS-CoV-2 can trigger complement activation, the pathways that are involved and the functional significance of the resultant effect. Methods: SARS-CoV-2 was inoculated into a human lepirudin-anticoagulated whole blood model, which contains a full repertoire of complement factors and leukocytes that express complement receptors. Complement activation was determined by measuring C5a production with an ELISA, and pretreatment with specific inhibitors was used to identify the pathways involved. The functional significance of this was then assessed by measuring markers of C5a signalling including leukocyte C5aR1 internalisation and CD11b upregulation with flow cytometry. Results: SARS-CoV-2 inoculation in this whole blood model caused progressive C5a production over 24 h, which was significantly reduced by inhibitors for factor B, C3, C5 and heparan sulfate. However, this phenomenon could not be replicated in cell-free plasma, highlighting the requirement for cell surface interactions with heparan sulfate. Functional analysis of this phenomenon revealed that C5aR1 signalling and CD11b upregulation in granulocytes and monocytes was delayed and only occurred after 24 h. Conclusion: SARS-CoV-2 is a noncanonical alternative pathway activator that progressively triggers complement activation through interactions with heparan sulfate.

Skin-patch delivered subunit vaccine induces broadly neutralising antibodies against SARS-CoV-2 variants of concern.

The ongoing SARS-CoV-2 pandemic continues to pose an enormous health challenge globally. The ongoing emergence of variants of concern has resulted in decreased vaccine efficacy necessitating booster immunizations. This was particularly highlighted by the recent emergence of the Omicron variant, which contains over 30 mutations in the spike protein and quickly became the dominant viral strain in global circulation. We previously demonstrated that delivery of a SARS-CoV-2 subunit vaccine via a high-density microarray patch (HD-MAP) induced potent immunity resulting in robust protection from SARS-CoV-2 challenge in mice. Here we show that serum from HD-MAP immunized animals maintained potent neutralisation against all variants tested, including Delta and Omicron. These findings highlight the advantages of HD-MAP vaccine delivery in inducing high levels of neutralising antibodies and demonstrates its potential at providing protection from emerging viral variants.

Nucleocapsid Specific Diagnostics for the Detection of Divergent SARS-CoV-2 Variants.

Since the start of the COVID-19 pandemic, multiple waves of SARS-CoV-2 variants have emerged. Of particular concern is the omicron variant, which harbors 28 mutations in the spike glycoprotein receptor binding and N-terminal domains relative to the ancestral strain. The high mutability of SARS-CoV-2 therefore poses significant hurdles for development of universal assays that rely on spike-specific immune detection. To address this, more conserved viral antigens need to be targeted. In this work, we comprehensively demonstrate the use of nucleocapsid (N)-specific detection across several assays using previously described nanobodies C2 and E2. We show that these nanobodies are highly sensitive and can detect divergent SARS-CoV-2 ancestral, delta and omicron variants across several assays. By comparison, spike-specific antibodies S309 and CR3022 only disparately detect SARS-CoV-2 variant targets. As such, we conclude that N-specific detection could provide a standardized universal target for detection of current and emerging SARS-CoV-2 variants of concern.

Synthetic Heparan Sulfate Mimetic Pixatimod (PG545) Potently Inhibits SARS-CoV-2 by Disrupting the Spike-ACE2 Interaction.

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19.