A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein.

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used messenger RNA (mRNA) display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor-binding domain, N-terminal domain, and S2 region, distal to the angiotensin-converting enzyme 2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target.

Enhanced stability of the SARS CoV-2 spike glycoprotein following modification of an alanine cavity in the protein core.

The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein subunit and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3-4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity next to Ala1016 and Ala1020 in the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. S glycoprotein membrane fusion function was retained with Ala1016/Ala1020 cavity-filling mutations associated with improved recombinant S2P-FHA thermostability, however 2 mutants, A1016L and A1016V/A1020I, lacked ability to mediate entry of S-HIV-1 pseudoparticles into 293-ACE2 cells. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited neutralizing antibody with 50%-inhibitory dilutions (ID50s) in the range 2,700-5,110 for ancestral and Delta-derived viruses, and 210-1,744 for Omicron BA.1. The antigens elicited antibody specificities directed to the receptor-binding domain (RBD), N-terminal domain (NTD), fusion peptide and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S2P-FHA-like ectodomain oligomers in the absence of an external trimerization motif (T4 foldon), thus representing an alternative approach for stabilizing oligomeric S glycoprotein vaccines.

Immunoglobulin repertoire restriction characterizes the serological responses of patients with predominantly antibody deficiency.

BACKGROUND: Predominantly antibody deficiency (PAD) is the most common category of inborn errors of immunity and is underpinned by impaired generation of appropriate antibody diversity and quantity. In the clinic, responses are interrogated by assessment of vaccination responses, which is central to many PAD diagnoses. However, the composition of the generated antibody repertoire is concealed from traditional quantitative measures of serological responses. Leveraging modern mass spectrometry-based proteomics (MS-proteomics), it is possible to elaborate the molecular features of specific antibody repertoires, which may address current limitations of diagnostic vaccinology. OBJECTIVES: We sought to evaluate serum antibody responses in patients with PAD following vaccination with a neo-antigen (severe acute respiratory syndrome coronavirus-2 vaccination) using MS-proteomics. METHODS: Following severe acute respiratory syndrome coronavirus-2 vaccination, serological responses in individuals with PAD and healthy controls (HCs) were assessed by anti-S1 subunit ELISA and neutralization assays. Purified anti-S1 subunit IgG and IgM was profiled by MS-proteomics for IGHV subfamily usage and somatic hypermutation analysis. RESULTS: Twelve patients with PAD who were vaccine-responsive were recruited with 11 matched vaccinated HCs. Neutralization and end point anti-S1 titers were lower in PAD. All subjects with PAD demonstrated restricted anti-S1 IgG antibody repertoires, with usage of <5 IGHV subfamilies (median: 3; range 2-4), compared to ≥5 for the 11 HC subjects (P < .001). IGHV3-7 utilization was far less common in patients with PAD than in HCs (2 of 12 vs 10 of 11; P = .001). Amino acid substitutions due to somatic hypermutation per subfamily did not differ between groups. Anti-S1 IgM was present in 64% and 50% of HC and PAD cohorts, respectively, and did not differ significantly between HCs and patients with PAD. CONCLUSIONS: This study demonstrates the breadth of anti-S1 antibodies elicited by vaccination at the proteome level and identifies stereotypical restriction of IGHV utilization in the IgG repertoire in patients with PAD compared with HC subjects. Despite uniformly pauci-clonal antibody repertoires some patients with PAD generated potent serological responses, highlighting a possible limitation of traditional serological techniques. These findings suggest that IgG repertoire restriction is a key feature of antibody repertoires in PAD.

Upper Respiratory Tract OC43 Infection Model for Investigating Airway Immune-Modifying Therapies.

Respiratory virus infections initiate and transmit from the upper respiratory tract (URT). Coronaviruses, including OC43, are a major cause of respiratory infection and disease. Failure to mount an effective antiviral immune response in the nasal mucosa increases the risk of severe disease and person-to-person transmission, highlighting the need for URT infection models to support the development of nasal treatments that improve coronavirus antiviral immunity. We aimed to determine if OC43 productively infected the mouse URT and would therefore be a suitable model to assess the efficacy and mechanism of action of nasal-targeting immune-modifying treatments. We administered OC43 via intranasal inoculation to wild-type Balb/c mice and assessed virus airway tropism (by comparing total respiratory tract vs. URT-only virus exposure) and characterized infection-induced immunity by quantifying specific antiviral cytokines and performing gene array assessment of immune genes. We then assessed the effect of immune-modulating therapies, including an immune-stimulating TLR2/6 agonist (INNA-X) and the immune-suppressing corticosteroid fluticasone propionate (FP). OC43 replicated in nasal respiratory epithelial cells, with peak viral RNA observed 2 days after infection. Prophylactic treatment with INNA-X accelerated expression of virus-induced IFN-λ and IFN-stimulated genes. In contrast, intranasal FP treatment increased nasal viral load by 2.4 fold and inhibited virus-induced IFN and IFN-stimulated gene expression. Prior INNA-X treatment reduced the immune-suppressive effect of FP. We demonstrate that the mouse nasal epithelium is permissive to OC43 infection and strengthen the evidence that TLR2 activation is a ß-coronavirus innate immune determinant and therapeutic target.

Infection and Vaccine Induced Spike Antibody Responses Against SARS-CoV-2 Variants of Concern in COVID-19-Naïve Children and Adults.

Although a more efficient adaptive humoral immune response has been proposed to underlie the usually favorable outcome of pediatric COVID-19, the breadth of viral and vaccine cross-reactivity toward the ever-mutating Spike protein among variants of concern (VOCs) has not yet been compared between children and adults. We assessed antibodies to conformational Spike in COVID-19-naïve children and adults vaccinated by BNT162b2 and ChAdOx1, and naturally infected with SARS-CoV-2 Early Clade, Delta, and Omicron. Sera were analyzed against Spike including naturally occurring VOCs Alpha, Beta, Gamma, Delta, and Omicron BA.1, BA.2, BA.5, BQ.1.1, BA2.75.2, and XBB.1, and variants of interest Epsilon, Kappa, Eta, D.2, and artificial mutant Spikes. There was no notable difference between breadth and longevity of antibody against VOCs in children and adults. Vaccinated individuals displayed similar immunoreactivity profiles across variants compared with naturally infected individuals. Delta-infected patients had an enhanced cross-reactivity toward Delta and earlier VOCs compared to patients infected by Early Clade SARS-CoV-2. Although Omicron BA.1, BA.2, BA.5, BQ.1.1, BA2.75.2, and XBB.1 antibody titers were generated after Omicron infection, cross-reactive binding against Omicron subvariants was reduced across all infection, immunization, and age groups. Some mutations, such as 498R and 501Y, epistatically combined to enhance cross-reactive binding, but could not fully compensate for antibody-evasive mutations within the Omicron subvariants tested. Our results reveal important molecular features central to the generation of high antibody titers and broad immunoreactivity that should be considered in future vaccine design and global serosurveillance in the context of limited vaccine boosters available to the pediatric population.

SARS-CoV-2 neutralizing antibodies: Longevity, breadth, and evasion by emerging viral variants.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antibody neutralization response and its evasion by emerging viral variants and variant of concern (VOC) are unknown, but critical to understand reinfection risk and breakthrough infection following vaccination. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus-cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 reverse transcription polymerase chain reaction (RT-PCR)-confirmed Coronavirus Disease 2019 (COVID-19) individuals with detailed demographics and followed up to 7 months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization, was associated with COVID-19 severity. A subgroup of "high responders" maintained high neutralizing responses over time, representing ideal convalescent plasma donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants and VOC. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal responders and vaccine monitoring and design.

Rapid HIV-1 Capsid Interaction Screening Using Fluorescence Fluctuation Spectroscopy.

The HIV capsid is a multifunctional protein capsule that mediates the delivery of the viral genetic material into the nucleus of the target cell. Host cell proteins bind to a number of repeating binding sites on the capsid to regulate steps in the replication cycle. Here, we develop a fluorescence fluctuation spectroscopy method using self-assembled capsid particles as the bait to screen for fluorescence-labeled capsid-binding analytes ("prey" molecules) in solution. The assay capitalizes on the property of the HIV capsid as a multivalent interaction platform, facilitating high sensitivity detection of multiple prey molecules that have accumulated onto capsids as spikes in fluorescence intensity traces. By using a scanning stage, we reduced the measurement time to 10 s without compromising on sensitivity, providing a rapid binding assay for screening libraries of potential capsid interactors. The assay can also identify interfaces for host molecule binding by using capsids with defects in known interaction interfaces. Two-color coincidence detection using the fluorescent capsid as the bait further allows the quantification of binding levels and determination of binding affinities. Overall, the assay provides new tools for the discovery and characterization of molecules used by the HIV capsid to orchestrate infection. The measurement principle can be extended for the development of sensitive interaction assays, utilizing natural or synthetic multivalent scaffolds as analyte-binding platforms.

HIV infection is influenced by dynamin at 3 independent points in the viral life cycle.

CD4 T cells are important cellular targets for HIV-1, yet the primary site of HIV fusion remains unresolved. Candidate fusion sites are either the plasma membrane or from within endosomes. One area of investigation compounding the controversy of this field, is the role of the protein dynamin in the HIV life cycle. To understand the role of dynamin in primary CD4 T cells we combined dynamin inhibition with a series of complementary assays based on single particle tracking, HIV fusion, detection of HIV DNA products and active viral transcription. We identify 3 levels of dynamin influence on the HIV life cycle. Firstly, dynamin influences productive infection by preventing cell cycle progression. Secondly, dynamin influences endocytosis rates and increases the probability of endosomal fusion. Finally, we provide evidence in resting CD4 T cells that dynamin directly regulates the HIV fusion reaction at the plasma membrane. We confirm this latter observation using 2 divergent dynamin modulating compounds, one that enhances dynamin conformations associated with dynamin ring formation (ryngo-1-23) and the other that preferentially targets dynamin conformations that appear in helices (dyngo-4a). This in-depth understanding of dynamin's roles in HIV infection clarifies recent controversies and furthermore provides evidence for dynamin regulation specifically in the HIV fusion reaction.

Dynamic imaging of the hepatitis C virus NS5A protein during a productive infection.

UNLABELLED: Hepatitis C virus (HCV) NS5A is essential for viral genome replication within cytoplasmic replication complexes and virus assembly at the lipid droplet (LD) surface, although its definitive functions are poorly understood. We developed approaches to investigate NS5A dynamics during a productive infection. We report here that NS5A motility and efficient HCV RNA replication require the microtubule network and the cytoplasmic motor dynein and demonstrate that both motile and relatively static NS5A-positive foci are enriched with host factors VAP-A and Rab5A. Pulse-chase imaging revealed that newly synthesized NS5A foci are small and distinct from aged foci, while further studies using a unique dual fluorescently tagged infectious HCV chimera showed a relatively stable association of NS5A foci with core-capped LDs. These results reveal new details about the dynamics and maturation of NS5A and the nature of potential sites of convergence of HCV replication and assembly pathways. IMPORTANCE: Hepatitis C virus (HCV) is a major cause of serious liver disease worldwide. An improved understanding of the HCV replication cycle will enable development of novel and improved antiviral strategies. Here we have developed complementary fluorescent labeling and imaging approaches to investigate the localization, traffic and interactions of the HCV NS5A protein in living, virus-producing cells. These studies reveal new details as to the traffic, composition and biogenesis of NS5A foci and the nature of their association with putative sites of virus assembly.

The microvesicle component of HIV-1 inocula modulates dendritic cell infection and maturation and enhances adhesion to and activation of T lymphocytes.

HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs) into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level de novo infection that can also be transferred to T lymphocytes at a later stage. We have previously reported that HIV-1 can induce partial maturation of iMDDCs at both stages of trafficking. Here we show that CD45⁺ microvesicles (MV) which contaminate purified HIV-1 inocula due to similar size and density, affect DC maturation, de novo HIV-1 infection and transfer to T lymphocytes. Comparing iMDDCs infected with CD45-depleted HIV-1BaL or matched non-depleted preparations, the presence of CD45⁺ MVs was shown to enhance DC maturation and ICAM-1 (CD54) expression, which is involved in DC∶T lymphocyte interactions, while restricting HIV-1 infection of MDDCs. Furthermore, in the DC culture HIV-1 infected (p24⁺) MDDCs were more mature than bystander cells. Depletion of MVs from the HIV-1 inoculum markedly inhibited DC∶T lymphocyte clustering and the induction of alloproliferation as well as limiting HIV-1 transfer from DCs to T lymphocytes. The effects of MV depletion on these functions were reversed by the re-addition of purified MVs from activated but not non-activated SUPT1.CCR5-CL.30 or primary T cells. Analysis of the protein complement of these MVs and of these HIV-1 inocula before and after MV depletion showed that Heat Shock Proteins (HSPs) and nef were the likely DC maturation candidates. Recombinant HSP90α and ß and nef all induced DC maturation and ICAM-1 expression, greater when combined. These results suggest that MVs contaminating HIV-1 released from infected T lymphocytes may be biologically important, especially in enhancing T cell activation, during uptake by DCs in vitro and in vivo, particularly as MVs have been detected in the circulation of HIV-1 infected subjects.

Mobilization of HIV spread by diaphanous 2 dependent filopodia in infected dendritic cells.

Paramount to the success of persistent viral infection is the ability of viruses to navigate hostile environments en route to future targets. In response to such obstacles, many viruses have developed the ability of establishing actin rich-membrane bridges to aid in future infections. Herein through dynamic imaging of HIV infected dendritic cells, we have observed how viral high-jacking of the actin/membrane network facilitates one of the most efficient forms of HIV spread. Within infected DC, viral egress is coupled to viral filopodia formation, with more than 90% of filopodia bearing immature HIV on their tips at extensions of 10 to 20 µm. Live imaging showed HIV filopodia routinely pivoting at their base, and projecting HIV virions at µm.sec⁻¹ along repetitive arc trajectories. HIV filopodial dynamics lead to up to 800 DC to CD4 T cell contacts per hour, with selection of T cells culminating in multiple filopodia tethering and converging to envelope the CD4 T-cell membrane with budding HIV particles. Long viral filopodial formation was dependent on the formin diaphanous 2 (Diaph2), and not a dominant Arp2/3 filopodial pathway often associated with pathogenic actin polymerization. Manipulation of HIV Nef reduced HIV transfer 25-fold by reducing viral filopodia frequency, supporting the potency of DC HIV transfer was dependent on viral filopodia abundance. Thus our observations show HIV corrupts DC to CD4 T cell interactions by physically embedding at the leading edge contacts of long DC filopodial networks.

HIV-1 infection of human macrophages directly induces viperin which inhibits viral production.

Macrophages are key target cells for HIV-1. HIV-1(BaL) induced a subset of interferon-stimulated genes in monocyte-derived macrophages (MDMs), which differed from that in monocyte-derived dendritic cells and CD4 T cells, without inducing any interferons. Inhibition of type I interferon induction was mediated by HIV-1 inhibition of interferon-regulated factor (IRF3) nuclear translocation. In MDMs, viperin was the most up-regulated interferon-stimulated genes, and it significantly inhibited HIV-1 production. HIV-1 infection disrupted lipid rafts via viperin induction and redistributed viperin to CD81 compartments, the site of HIV-1 egress by budding in MDMs. Exogenous farnesol, which enhances membrane protein prenylation, reversed viperin-mediated inhibition of HIV-1 production. Mutagenesis analysis in transfected cell lines showed that the internal S-adenosyl methionine domains of viperin were essential for its antiviral activity. Thus viperin may contribute to persistent noncytopathic HIV-1 infection of macrophages and possibly to biologic differences with HIV-1-infected T cells.

Rapid spread of the SARS-CoV-2 JN.1 lineage is associated with increased neutralization evasion.

In July/August 2023, the highly mutated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) BA.2.86 lineage emerged and its descendant JN.1 is on track to become the dominant SARS-CoV-2 lineage globally. Compared to the spike (S) protein of the parental BA.2.86 lineage, the JN.1 S protein contains one mutation, L455S, which may affect receptor binding and antibody evasion. Here, we performed a virological assessment of the JN.1 lineage employing pseudovirus particles bearing diverse SARS-CoV-2 S proteins. Using this strategy, it was found that S protein mutation L455S confers increased neutralization resistance but reduces ACE2 binding capacity and S protein-driven cell entry efficiency. Altogether, these data suggest that the benefit of increased antibody evasion outweighs the reduced ACE2 binding capacity and further enabled the JN.1 lineage to effectively spread in the human population.

Pharmacologic hyperstabilisation of the HIV-1 capsid lattice induces capsid failure.

The HIV-1 capsid has emerged as a tractable target for antiretroviral therapy. Lenacapavir, developed by Gilead Sciences, is the first capsid-targeting drug approved for medical use. Here, we investigate the effect of lenacapavir on HIV capsid stability and uncoating. We employ a single particle approach that simultaneously measures capsid content release and lattice persistence. We demonstrate that lenacapavir's potent antiviral activity is predominantly due to lethal hyperstabilisation of the capsid lattice and resultant loss of compartmentalisation. This study highlights that disrupting capsid metastability is a powerful strategy for the development of novel antivirals.

Improvement of immune dysregulation in individuals with long COVID at 24-months following SARS-CoV-2 infection.

This study investigates the humoral and cellular immune responses and health-related quality of life measures in individuals with mild to moderate long COVID (LC) compared to age and gender matched recovered COVID-19 controls (MC) over 24 months. LC participants show elevated nucleocapsid IgG levels at 3 months, and higher neutralizing capacity up to 8 months post-infection. Increased spike-specific and nucleocapsid-specific CD4+ T cells, PD-1, and TIM-3 expression on CD4+ and CD8+ T cells were observed at 3 and 8 months, but these differences do not persist at 24 months. Some LC participants had detectable IFN-γ and IFN-ß, that was attributed to reinfection and antigen re-exposure. Single-cell RNA sequencing at the 24 month timepoint shows similar immune cell proportions and reconstitution of naïve T and B cell subsets in LC and MC. No significant differences in exhaustion scores or antigen-specific T cell clones are observed. These findings suggest resolution of immune activation in LC and return to comparable immune responses between LC and MC over time. Improvement in self-reported health-related quality of life at 24 months was also evident in the majority of LC (62%). PTX3, CRP levels and platelet count are associated with improvements in health-related quality of life.

Revising the Role of Myeloid cells in HIV Pathogenesis.

Lentiviruses are characterized by their ability to infect resting cells, such as CD4 T cells, macrophages and dendritic cells (DC). Cells of myeloid lineage, which herein we include including monocytes, macrophages, and dendritic cells, play a pivotal role in HIV infection by not only promoting transmission and spread but also serving as viral reservoirs. However, the recent discovery of the HIV restriction factor SAMHD1 within myeloid cells has again led us to question the role of this lineage both in HIV transmission and pathogenesis. Herein we will summarize what the potential role of myeloid cells in HIV pathogenesis is and how recent observations have or haven't reshaped this view. Finally we highlight the idea that cells of myeloid lineage are quality rather than quantity HIV substrates. Thus, whilst is may indeed be difficult for a lentivirus like HIV to infect a resting cell like a macrophage and/or Dendritic cell, there are significant benefits in doing so, even at low frequency.

Durable reprogramming of neutralizing antibody responses following Omicron breakthrough infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) breakthrough infection of vaccinated individuals is increasingly common with the circulation of highly immune evasive and transmissible Omicron variants. Here, we report the dynamics and durability of recalled spike-specific humoral immunity following Omicron BA.1 or BA.2 breakthrough infection, with longitudinal sampling up to 8 months after infection. Both BA.1 and BA.2 infections robustly boosted neutralization activity against the infecting strain while expanding breadth against BA.4, although neutralization activity was substantially reduced for the more recent XBB and BQ.1.1 strains. Cross-reactive memory B cells against both ancestral and Omicron spike were predominantly expanded by infection, with limited recruitment of de novo Omicron-specific B cells or antibodies. Modeling of neutralization titers predicts that protection from symptomatic reinfection against antigenically similar strains will be durable but is undermined by new emerging strains with further neutralization escape.

Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients.

Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells of convalescent patients using SARS-CoV-2 receptor binding domains carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (S309) by orders of magnitude. They also provide prophylactic and therapeutic in vivo protection of female hACE2 mice against viral challenge. Our results indicate that exposure to SARS-CoV-2 induces antibodies that maintain broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.

Emergence and antibody evasion of BQ, BA.2.75 and SARS-CoV-2 recombinant sub-lineages in the face of maturing antibody breadth at the population level.

BACKGROUND: The Omicron era of the COVID-19 pandemic commenced at the beginning of 2022 and whilst it started with primarily BA.1, it was latter dominated by BA.2 and the related sub-lineage BA.5. Following resolution of the global BA.5 wave, a diverse grouping of Omicron sub-lineages emerged derived from BA.2, BA.5 and recombinants thereof. Whilst emerging from distinct lineages, all shared similar changes in the Spike glycoprotein affording them an outgrowth advantage through evasion of neutralising antibodies. METHODS: Over the course of 2022, we monitored the potency and breadth of antibody neutralization responses to many emerging variants in the Australian community at three levels: (i) we tracked over 420,000 U.S. plasma donors over time through various vaccine booster roll outs and Omicron waves using sequentially collected IgG pools; (ii) we mapped the antibody response in individuals using blood from stringently curated vaccine and convalescent cohorts. (iii) finally we determine the in vitro efficacy of clinically approved therapies Evusheld and Sotrovimab. FINDINGS: In pooled IgG samples, we observed the maturation of neutralization breadth to Omicron variants over time through continuing vaccine and infection waves. Importantly, in many cases, we observed increased antibody breadth to variants that were yet to be in circulation. Determination of viral neutralization at the cohort level supported equivalent coverage across prior and emerging variants with isolates BQ.1.1, XBB.1, BR.2.1 and XBF the most evasive. Further, these emerging variants were resistant to Evusheld, whilst increasing neutralization resistance to Sotrovimab was restricted to BQ.1.1 and XBF. We conclude at this current point in time that dominant variants can evade antibodies at levels equivalent to their most evasive lineage counterparts but sustain an entry phenotype that continues to promote an additional outgrowth advantage. In Australia, BR.2.1 and XBF share this phenotype and, in contrast to global variants, are uniquely dominant in this region in the later months of 2022. INTERPRETATION: Whilst the appearance of a diverse range of omicron lineages has led to primary or partial resistance to clinically approved monoclonal antibodies, the maturation of the antibody response across both cohorts and a large donor pools importantly observes increasing breadth in the antibody neutralisation responses over time with a trajectory that covers both current and known emerging variants. FUNDING: This work was primarily supported by Australian Medical Foundation research grants MRF2005760 (SGT, GM & WDR), Medical Research Future Fund Antiviral Development Call grant (WDR), the New South Wales Health COVID-19 Research Grants Round 2 (SGT & FB) and the NSW Vaccine Infection and Immunology Collaborative (VIIM) (ALC). Variant modeling was supported by funding from SciLifeLab's Pandemic Laboratory Preparedness program to B.M. (VC-2022-0028) and by the European Union's Horizon 2020 research and innovation programme under grant agreement no. 101003653 (CoroNAb) to B.M.