Herpes simplex virus spreads rapidly in human foreskin, partly driven by chemokine-induced redistribution of Nectin-1 on keratinocytes.

HSV infects keratinocytes in the epidermis of skin via nectin-1. We established a human foreskin explant infection model to investigate HSV entry and spread. HSV1 entry could only be achieved by the topical application of virus via high density microarray projections (HD-MAPs) to the epidermis, which penetrated beyond one third of its thickness, simulating in vivo microtrauma. Rapid lateral spread of HSV1 to a mean of 13 keratinocytes wide occurred after 24 hours and free virus particles were observed between keratinocytes, consistent with an intercellular route of spread. Nectin-1 staining was markedly decreased in foci of infection in the epidermis and in the human keratinocyte HaCaT cell line. Nectin-1 was redistributed, at the protein level, in adjacent uninfected cells surrounding infection, inducible by CCL3, IL-8 (or CXCL8), and possibly CXCL10 and IL-6, thus facilitating spread. These findings provide the first insights into HSV1 entry and spread in human inner foreskin in situ.

Characterising plasmacytoid and myeloid AXL+ SIGLEC-6+ dendritic cell functions and their interactions with HIV.

AXL+ Siglec-6+ dendritic cells (ASDC) are novel myeloid DCs which can be subdivided into CD11c+ and CD123+ expressing subsets. We showed for the first time that these two ASDC subsets are present in inflamed human anogenital tissues where HIV transmission occurs. Their presence in inflamed tissues was supported by single cell RNA analysis of public databases of such tissues including psoriasis diseased skin and colorectal cancer. Almost all previous studies have examined ASDCs as a combined population. Our data revealed that the two ASDC subsets differ markedly in their functions when compared with each other and to pDCs. Relative to their cell functions, both subsets of blood ASDCs but not pDCs expressed co-stimulatory and maturation markers which were more prevalent on CD11c+ ASDCs, thus inducing more T cell proliferation and activation than their CD123+ counterparts. There was also a significant polarisation of naïve T cells by both ASDC subsets toward Th2, Th9, Th22, Th17 and Treg but less toward a Th1 phenotype. Furthermore, we investigated the expression of chemokine receptors that facilitate ASDCs and pDCs migration from blood to inflamed tissues, their HIV binding receptors, and their interactions with HIV and CD4 T cells. For HIV infection, within 2 hours of HIV exposure, CD11c+ ASDCs showed a trend in more viral transfer to T cells than CD123+ ASDCs and pDCs for first phase transfer. However, for second phase transfer, CD123+ ASDCs showed a trend in transferring more HIV than CD11c+ ASDCs and there was no viral transfer from pDCs. As anogenital inflammation is a prerequisite for HIV transmission, strategies to inhibit ASDC recruitment into inflamed tissues and their ability to transmit HIV to CD4 T cells should be considered.

Multiple COVID-19 vaccine doses in CLL and MBL improve immune responses with progressive and high seroconversion.

Patients with chronic lymphocytic leukemia (CLL) or monoclonal B-lymphocytosis (MBL) have impaired response to COVID-19 vaccination. A total of 258 patients (215 with CLL and 43 with MBL) had antispike antibody levels evaluable for statistical analysis. The overall seroconversion rate in patients with CLL was 94.2% (antispike antibodies ≥50 AU/mL) and 100% in patients with MBL after multiple vaccine doses. After 3 doses (post-D3) in 167 patients with CLL, 73.7% were seropositive, 17.4% had antispike antibody levels between 50 and 999 AU/mL, and 56.3% had antispike antibody levels ≥1000 AU/mL, with a median rise from 144.6 to 1800.7 AU/mL. Of patients who were seronegative post-D2, 39.7% seroconverted post-D3. For those who then remained seronegative after their previous dose, seroconversion occurred in 40.6% post-D4, 46.2% post-D5, 16.7% post-D6, and 0% after D7 or D8. After seroconversion, most had a progressive increase in antispike antibody levels. Neutralization was associated with higher antispike antibody levels, more vaccine doses, and earlier severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants; neutralizing antibody against early clade D614G was detected in 65.3%, against Delta in 52.0%, and against Omicron in 36.5%. SARS-CoV-2-specific T-cell production of interferon γ and interleukin 2 occurred in 73.9% and 60.9%, respectively, of 23 patients tested. After multiple vaccine doses, by multivariate analysis, immunoglobulin M ≥0.53 g/L, immunoglobulin subclass G3 ≥0.22 g/L and absence of current CLL therapy were independent predictors of positive serological responses. Multiple sequential COVID-19 vaccination significantly increased seroconversion and antispike antibody levels in patients with CLL or MBL.

Herpes Simplex Virus type 1 infects Langerhans cells and the novel epidermal dendritic cell, Epi-cDC2s, via different entry pathways.

Skin mononuclear phagocytes (MNPs) provide the first interactions of invading viruses with the immune system. In addition to Langerhans cells (LCs), we recently described a second epidermal MNP population, Epi-cDC2s, in human anogenital epidermis that is closely related to dermal conventional dendritic cells type 2 (cDC2) and can be preferentially infected by HIV. Here we show that in epidermal explants topically infected with herpes simplex virus (HSV-1), both LCs and Epi-cDC2s interact with HSV-1 particles and infected keratinocytes. Isolated Epi-cDC2s support higher levels of infection than LCs in vitro, inhibited by acyclovir, but both MNP subtypes express similar levels of the HSV entry receptors nectin-1 and HVEM, and show similar levels of initial uptake. Using inhibitors of endosomal acidification, actin and cholesterol, we found that HSV-1 utilises different entry pathways in each cell type. HSV-1 predominantly infects LCs, and monocyte-derived MNPs, via a pH-dependent pathway. In contrast, Epi-cDC2s are mainly infected via a pH-independent pathway which may contribute to the enhanced infection of Epi-cDC2s. Both cells underwent apoptosis suggesting that Epi-cDC2s may follow the same dermal migration and uptake by dermal MNPs that we have previously shown for LCs. Thus, we hypothesize that the uptake of HSV and infection of Epi-cDC2s will stimulate immune responses via a different pathway to LCs, which in future may help guide HSV vaccine development and adjuvant targeting.

COVID-19 vaccine failure in chronic lymphocytic leukaemia and monoclonal B-lymphocytosis; humoural and cellular immunity.

Chronic lymphocytic leukaemia (CLL) is associated with immunocompromise and high risk of severe COVID-19 disease and mortality. Monoclonal B-cell lymphocytosis (MBL) patients also have immune impairment. We evaluated humoural and cellular immune responses in 181 patients with CLL (160) and MBL (21) to correlate failed seroconversion [<50 AU/ml SARS-CoV-2 II IgG assay, antibody to spike protein; Abbott Diagnostics)] following each of two vaccine doses with clinical and laboratory parameters. Following first and second doses, 79.2% then 45% of CLL, and 50% then 9.5% of MBL patients respectively remained seronegative. There was significant association between post dose two antibody level with pre-vaccination reduced IgM (p < 0.0001), IgG2 (p < 0.035), and IgG3 (p < 0.046), and CLL therapy within 12 months (p < 0.001) in univariate analysis. By multivariate analysis, reduced IgM (p < 0.0002) and active therapy (p < 0.0002) retained significance. Anti-spike protein levels varied widely and were lower in CLL than MBL patients, and both lower than in normal donors. Neutralisation activity showed anti-spike levels <1000 AU/ml were usually negative for both an early viral clade and the contemporary Delta variant and 72.9% of CLL and 53.3% of MBL failed to reach levels ≥1000 AU/ml. In a representative sample, ~80% had normal T-cell responses. Failed seroconversion occurred in 36.6% of treatment-naïve patients, in 78.1% on therapy, and in 85.7% on ibrutinib.

Identification of SARS-CoV-2 Nucleocapsid and Spike T-Cell Epitopes for Assessing T-Cell Immunity.

Developing optimal T-cell response assays to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is critical for measuring the duration of immunity to this disease and assessing the efficacy of vaccine candidates. These assays need to target conserved regions of SARS-CoV-2 global variants and avoid cross-reactivity to seasonal human coronaviruses. To contribute to this effort, we employed an in silico immunoinformatics analysis pipeline to identify immunogenic peptides resulting from conserved and highly networked regions with topological importance from the SARS-CoV-2 nucleocapsid and spike proteins. A total of 57 highly networked T-cell epitopes that are conserved across geographic viral variants were identified from these viral proteins, with a binding potential to diverse HLA alleles and 80 to 100% global population coverage. Importantly, 18 of these T-cell epitope derived peptides had limited homology to seasonal human coronaviruses making them promising candidates for SARS-CoV-2-specific T-cell immunity assays. Moreover, two of the NC-derived peptides elicited effector/polyfunctional responses of CD8+ T cells derived from SARS-CoV-2 convalescent patients.IMPORTANCE The development of specific and validated immunologic tools is critical for understanding the level and duration of the cellular response induced by SARS-CoV-2 infection and/or vaccines against this novel coronavirus disease. To contribute to this effort, we employed an immunoinformatics analysis pipeline to define 57 SARS-CoV-2 immunogenic peptides within topologically important regions of the nucleocapsid (NC) and spike (S) proteins that will be effective for detecting cellular immune responses in 80 to 100% of the global population. Our immunoinformatics analysis revealed that 18 of these peptides had limited homology to circulating seasonal human coronaviruses and therefore are promising candidates for distinguishing SARS-CoV-2-specific immune responses from pre-existing coronavirus immunity. Importantly, CD8+ T cells derived from SARS-CoV-2 survivors exhibited polyfunctional effector responses to two novel NC-derived peptides identified as HLA-binders. These studies provide a proof of concept that our immunoinformatics analysis pipeline identifies novel immunogens which can elicit polyfunctional SARS-CoV-2-specific T-cell responses.

AFid: a tool for automated identification and exclusion of autofluorescent objects from microscopy images.

MOTIVATION: Autofluorescence is a long-standing problem that has hindered the analysis of images of tissues acquired by fluorescence microscopy. Current approaches to mitigate autofluorescence in tissue are lab-based and involve either chemical treatment of sections or specialized instrumentation and software to 'unmix' autofluorescent signals. Importantly, these approaches are pre-emptive and there are currently no methods to deal with autofluorescence in acquired fluorescence microscopy images. RESULTS: To address this, we developed Autofluorescence Identifier (AFid). AFid identifies autofluorescent pixels as discrete objects in multi-channel images post-acquisition. These objects can then be tagged for exclusion from downstream analysis. We validated AFid using images of FFPE human colorectal tissue stained for common immune markers. Further, we demonstrate its utility for image analysis where its implementation allows the accurate measurement of HIV-Dendritic cell interactions in a colorectal explant model of HIV transmission. Therefore, AFid represents a major leap forward in the extraction of useful data from images plagued by autofluorescence by offering an approach that is easily incorporated into existing workflows and that can be used with various samples, staining panels and image acquisition methods. We have implemented AFid in ImageJ, Matlab and R to accommodate the diverse image analysis community. AVAILABILITY AND IMPLEMENTATION: AFid software is available at https://ellispatrick.github.io/AFid. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Relay of herpes simplex virus between Langerhans cells and dermal dendritic cells in human skin.

The mechanism by which immunity to Herpes Simplex Virus (HSV) is initiated is not completely defined. HSV initially infects mucosal epidermis prior to entering nerve endings. In mice, epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to encounter HSV, but it is CD103(+) dermal DCs that carry viral antigen to lymph nodes for antigen presentation, suggesting DC cross-talk in skin. In this study, we compared topically HSV-1 infected human foreskin explants with biopsies of initial human genital herpes lesions to show LCs are initially infected then emigrate into the dermis. Here, LCs bearing markers of maturation and apoptosis formed large cell clusters with BDCA3(+) dermal DCs (thought to be equivalent to murine CD103(+) dermal DCs) and DC-SIGN(+) DCs/macrophages. HSV-expressing LC fragments were observed inside the dermal DCs/macrophages and the BDCA3(+) dermal DCs had up-regulated a damaged cell uptake receptor CLEC9A. No other infected epidermal cells interacted with dermal DCs. Correspondingly, LCs isolated from human skin and infected with HSV-1 in vitro also underwent apoptosis and were taken up by similarly isolated BDCA3(+) dermal DCs and DC-SIGN(+) cells. Thus, we conclude a viral antigen relay takes place where HSV infected LCs undergo apoptosis and are taken up by dermal DCs for subsequent antigen presentation. This provides a rationale for targeting these cells with mucosal or perhaps intradermal HSV immunization.

Human Anti-CD40 Antibody and Poly IC:LC Adjuvant Combination Induces Potent T Cell Responses in the Lung of Nonhuman Primates.

Nonlive vaccine platforms that induce potent cellular immune responses in mucosal tissue would have broad application for vaccines against infectious diseases and tumors. Induction of cellular immunity could be optimized by targeted activation of multiple innate and costimulatory signaling pathways, such as CD40 or TLRs. In this study, we evaluated immune activation and elicitation of T cell responses in nonhuman primates after immunization with peptide Ags adjuvanted with an agonistic anti-CD40Ab, with or without the TLR3 ligand poly IC:LC. We found that i.v. administration of the anti-CD40Ab induced rapid and transient innate activation characterized by IL-12 production and upregulated costimulatory and lymph node homing molecules on dendritic cells. Using fluorescently labeled Abs for in vivo tracking, we found that the anti-CD40Ab bound to all leukocytes, except T cells, and disseminated to multiple organs. CD4(+) and CD8(+) T cell responses were significantly enhanced when the anti-CD40Ab was coadministered with poly IC:LC compared with either adjuvant given alone and were almost exclusively compartmentalized to the lung. Notably, Ag-specific T cells in the bronchoalveolar lavage were sustained at ∼5-10%. These data indicate that systemic administration of anti-CD40Ab may be particularly advantageous for vaccines and/or therapies that require T cell immunity in the lung.

Protection of human myeloid dendritic cell subsets against influenza A virus infection is differentially regulated upon TLR stimulation.

The proinflammatory microenvironment in the respiratory airway induces maturation of both resident and infiltrating dendritic cells (DCs) upon influenza A virus (IAV) infection. This results in upregulation of antiviral pathways as well as modulation of endocytic processes, which affect the susceptibility of DCs to IAV infection. Therefore, it is highly relevant to understand how IAV interacts with and infects mature DCs. To investigate how different subsets of human myeloid DCs (MDCs) involved in tissue inflammation are affected by inflammatory stimulation during IAV infection, we stimulated primary blood MDCs and inflammatory monocyte-derived DCs (MDDCs) with TLR ligands, resulting in maturation. Interestingly, MDDCs but not MDCs were protected against IAV infection after LPS (TLR4) stimulation. In contrast, stimulation with TLR7/8 ligand protected MDCs but not MDDCs from IAV infection. The reduced susceptibility to IAV infection correlated with induction of type I IFNs. We found that differential expression of TLR4, TRIF, and MyD88 in the two MDC subsets regulated the ability of the cells to enter an antiviral state upon maturation. This difference was functionally confirmed using small interfering RNA and inhibitors. Our data show that different human MDC subsets may play distinct roles during IAV infection, as their capacity to induce type I IFNs is dependent on TLR-specific maturation, resulting in differential susceptibility to IAV infection.

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.

Identification of lineage relationships and novel markers of blood and skin human dendritic cells.

The lineage relationships and fate of human dendritic cells (DCs) have significance for a number of diseases including HIV where both blood and tissue DCs may be infected. We used gene expression profiling of human monocyte and DC subpopulations sorted directly from blood and skin to define the lineage relationships. We also compared these with monocyte-derived DCs (MDDCs) and MUTZ3 Langerhans cells (LCs) to investigate their relevance as model skin DCs. Hierarchical clustering analysis showed that myeloid DCs clustered according to anatomical origin rather than putative lineage. Plasmacytoid DCs formed the most discrete cluster, but ex vivo myeloid cells formed separate clusters of cells both in blood and in skin. Separate and specific DC populations could be determined within skin, and the proportion of CD14(+) dermal DCs (DDCs) was reduced and CD1a(+) DDCs increased during culture, suggesting conversion to CD1a(+)-expressing cells in situ. This is consistent with origin of the CD1a(+) DDCs from a local precursor rather than directly from circulating blood DCs or monocyte precursors. Consistent with their use as model skin DCs, the in vitro-derived MDDC and MUTZ3 LC populations grouped within the skin DC cluster. MDDCs clustered most closely to CD14(+) DDCs; furthermore, common unique patterns of C-type lectin receptor expression were identified between these two cell types. MUTZ3 LCs, however, did not cluster closely with ex vivo-derived LCs. We identified differential expression of novel genes in monocyte and DC subsets including genes related to DC surface receptors (including C-type lectin receptors, TLRs, and galectins).

Human plasmacytoid dendritic cells efficiently capture HIV-1 envelope glycoproteins via CD4 for antigen presentation.

Advances in HIV-1 vaccine clinical trials and preclinical research indicate that the virus envelope glycoproteins (Env) are likely to be an essential component of a prophylactic vaccine. Efficient Ag uptake and presentation by dendritic cells (DCs) is important for strong CD4(+) Th cell responses and the development of effective humoral immune responses. In this study, we examined the capacity of distinct primary human DC subsets to internalize and present recombinant Env to CD4(+) T cells. Consistent with their specific receptor expression, skin DCs bound and internalized Env via C-type lectin receptors, whereas blood DC subsets, including CD1c(+) myeloid DCs, CD123(+) plasmacytoid DCs (PDCs), and CD141(+) DCs exhibited a restricted repertoire of C-type lectin receptors and relied on CD4 for uptake of Env. Despite a generally poor capacity for Ag uptake compared with myeloid DCs, the high expression of CD4 on PDCs allowed them to bind and internalize Env very efficiently. CD4-mediated uptake delivered Env to EEA1(+) endosomes that progressed to Lamp1(+) and MHC class II(+) lysosomes where internalized Env was degraded rapidly. Finally, all three blood DC subsets were able to internalize an Env-CMV pp65 fusion protein via CD4 and stimulate pp65-specific CD4(+) T cells. Thus, in the in vitro systems described in this paper, CD4-mediated uptake of Env is a functional pathway leading to Ag presentation, and this may therefore be a mechanism used by blood DCs, including PDCs, for generating immune responses to Env-based vaccines.

Tissue dendritic cells as portals for HIV entry.

Dendritic cells (DCs) are found at the portals of pathogen entry such as the mucosal surfaces of the respiratory, gastrointestinal and genital tracts where they represent the first line of contact between the immune system and the foreign invaders. They are found throughout the body in multiple subsets where they express unique combinations of C-type lectin receptors to best aid them in detection of pathogens associated with their anatomical location. DCs are important in the establishment in HIV infection for two reasons. Firstly, they are one of the first cells to encounter the virus, and the specific interaction that occurs between these cells and HIV is critical to HIV establishing a foothold infection. Secondly and most importantly, HIV is able to efficiently transfer the virus to its primary target cell, the CD4(+) T lymphocyte, in which it replicates explosively. Infection of CD4(+) T lymphocytes via DCs is far more efficient than direct infection. This review surveys the various DCs subsets found within the human sexual mucosa and their interactions with HIV. Mechanisms of HIV uptake are discussed as well as how the virus then traffics through the DC and is transferred to T cells. Until recently, most research has focussed on vaginal transmission despite the increased transmission rate associated with anal intercourse. Here, we also discuss recent advances in our understanding of HIV transmission in the colon.

Advances in understanding the mechanism of action of adult vaccines.

The occurrence of herpes zoster (HZ) correlates with declining memory T cells that had responded to earlier infection with varicella-zoster virus (VZV). There are especially lower T cell responses to the single immunodominant VZV protein glycoprotein E (gE) in people over 50 years of age, although antibody responses to VZV persist. Therefore, a live attenuated zoster vaccine (ZVL) aimed at restoring T cell responses was developed. Surprisingly, a recombinant zoster vaccine (RZV) consisting of gE combined with the AS01B adjuvant system proved superior in efficacy and durability. In this issue of the JCI, Laing, Ford, and colleagues showed that both vaccines stimulated preimmunization naive CD4+ T cells, not just memory CD4+ T cells, to gE, and recruited these naive responses into the overall memory response. However, compared with ZVL, RZV stimulated this response to a much greater degree. These results will help guide development of more effective and durable vaccines for older individuals.

Highly Networked SARS-CoV-2 Peptides Elicit T Cell Responses with Enhanced Specificity.

Identifying SARS-CoV-2-specific T cell epitope-derived peptides is critical for the development of effective vaccines and measuring the duration of specific SARS-CoV-2 cellular immunity. In this regard, we previously identified T cell epitope-derived peptides within topologically and structurally essential regions of SARS-CoV-2 spike and nucleocapsid proteins by applying an immunoinformatics pipeline. In this study, we selected 30 spike- and nucleocapsid-derived peptides and assessed whether these peptides induce T cell responses and avoid major mutations found in SARS-CoV-2 variants of concern. Our peptide pool was highly specific, with only a single peptide driving cross-reactivity in people unexposed to SARS-COV-2, and immunogenic, inducing a polyfunctional response in CD4+ and CD8+ T cells from COVID-19 recovered individuals. All peptides were immunogenic and individuals recognized broad and diverse peptide repertoires. Moreover, our peptides avoided most mutations/deletions associated with all four SARS-CoV-2 variants of concern while retaining their physicochemical properties even when genetic changes are introduced. This study contributes to an evolving definition of individual CD4+ and CD8+ T cell epitopes that can be used for specific diagnostic tools for SARS-CoV-2 T cell responses and is relevant to the development of variant-resistant and durable T cell-stimulating vaccines.

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.

A differential role for macropinocytosis in mediating entry of the two forms of vaccinia virus into dendritic cells.

Vaccinia virus (VACV) is being developed as a recombinant viral vaccine vector for several key pathogens. Dendritic cells (DCs) are specialised antigen presenting cells that are crucial for the initiation of primary immune responses; however, the mechanisms of uptake of VACV by these cells are unclear. Therefore we examined the binding and entry of both the intracellular mature virus (MV) and extracellular enveloped virus (EV) forms of VACV into vesicular compartments of monocyte-derived DCs. Using a panel of inhibitors, flow cytometry and confocal microscopy we have shown that neither MV nor EV binds to the highly expressed C-type lectin receptors on DCs that are responsible for capturing many other viruses. We also found that both forms of VACV enter DCs via a clathrin-, caveolin-, flotillin- and dynamin-independent pathway that is dependent on actin, intracellular calcium and host-cell cholesterol. Both MV and EV entry were inhibited by the macropinocytosis inhibitors rottlerin and dimethyl amiloride and depended on phosphotidylinositol-3-kinase (PI(3)K), and both colocalised with dextran but not transferrin. VACV was not delivered to the classical endolysosomal pathway, failing to colocalise with EEA1 or Lamp2. Finally, expression of early viral genes was not affected by bafilomycin A, indicating that the virus does not depend on low pH to deliver cores to the cytoplasm. From these collective results we conclude that VACV enters DCs via macropinocytosis. However, MV was consistently less sensitive to inhibition and is likely to utilise at least one other entry pathway. Definition and future manipulation of these pathways may assist in enhancing the activity of recombinant vaccinia vectors through effects on antigen presentation.

Tissue resident memory T cells inhabit the deep human conjunctiva.

Mucosal linings of the body, including the conjunctiva, are enriched in tissue-resident memory T cells (TRMs) whose defining feature is their continual tissue protection that does not rely on migration to lymphoid organs to elicit immune responses. Hitherto, conjunctival TRMs have only been identified in the superficial epithelium. This work aims to develop a more complete understanding of the conjunctival immunological capacity by investigating the presence of TRMs within the deeper, more stable layers of the healthy human conjunctiva. Using immunofluorescence microscopy and antibodies against CD3, CD4, CD69 and HLA-DR on bulbar conjunctival biopsies obtained from 7 healthy adults (age range = 32-77 years; females = 4), we identified CD69+TRM subsets in all layers of the human conjunctiva: the superficial epithelium, the basal epithelium, the adenoid, and the fibrous layers. Interestingly, the adenoid layer showed significantly higher densities of both CD4 and CD8 TRMs when compared to the fibrous layer and conjunctival epithelia. Additionally, CD4 TRMs predominated significantly over CD8 TRMs in the adenoid layer. The abundance of deep conjunctival CD69+TRMs within the healthy human may suggest the presence of defence mechanisms capable of inducing long-term immunogenic memory. Understanding this spatial distribution of conjunctival CD69+TRMs is essential to improving mucosal vaccine design.