Anthony Dominic Kelleher

The yellow fever virus-17D (YFV-17D) vaccine is widely regarded as one of the most effective vaccines in existence, with protection linked to robust elicitation of neutralising antibodies ¹ . The features of the germinal centre (GC) reaction driving this durable and potent antibody responses remain unclear. Here we examined immune dynamics in blood and lymph nodes (LNs) after primary YFV-17D immunisation in humans. Using fine needle biopsy (FNB), we identified robust T and B lymphocyte responses within draining LNs, underpinning maturation of antibodies targeting the viral envelope (E). Early antibody responses were largely IgM, with gradual accumulation of E-specific memory B cells and anti-viral IgG in the subsequent months. Monoclonal antibodies derived from GC B cells had specificity for E, with diversification in the LN capable of driving increases in binding and neutralising potency. While circulating vaccine-specific CD4 ⁺ T cells peaked two weeks post-vaccination, clonally related GC and IL-10 ⁺ T follicular helper populations progressively accumulated in the LN. Comparison of FNB transcriptional datasets following YFV-17D, SARS-CoV-2 mRNA/LNP, and seasonal influenza vaccination identified a conserved GC program involving similar B and T lymphocyte subpopulations. Collectively, we find that while YFV-17D induces robust GC activity, this trait is shared with other vaccine platforms and the notably durable protection against infection may reflect an incapacity of the virus to escape host immunity.

Acute lung injury (ALI) is characterised by hypoxia and inflammation that derives from diverse aetiologies. Delineating the molecular pathogeneses may identify treatable subgroups and inform clinical trials. Using a multi-omics approach, we analysed trauma-associated ALI (n = 9), COVID-19 ALI (n = 11), ICU controls (n = 13) using scRNA-seq, serum cytokine profiling, and bronchoalveolar lavage (BAL) microbiome analysis. We identified a population of activated CD4⁺ T cells expressing Toll-like receptor 4 (TLR4) and downstream NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome complex, predominantly in trauma-associated ALI (23% of CD45⁺ Cells) compared to COVID-19 ALI (14%) and ICU controls (15%). Serum IL-1RA and IL-6 were upregulated in trauma ALI, along with TLR4-binding ligand hyaluronan. TLR4 expression on CD4⁺ T cells was confirmed by spectral cytometry. Lung microbiome analysis revealed distinct diversity in trauma-associated ALI compared to COVID-19 ALI. These findings highlight TLR4-mediated inflammation in trauma-associated ALI and suggest potential precision medicine approaches for ALI management.

The successive emergence of SARS-CoV-2 variants with altered tissue tropism has progressively decoupled transmissibility from lower respiratory tract pathogenicity. Omicron lineages transmit exceptionally well whilst limiting severe lung disease. Here, we demonstrate that this phenotype has evolved through two temporally distinct tissue-specific proteolytic controls at the Spike (S) furin cleavage site (FCS). At the virion level, cell-type-dependent furin-mediated FCS hyper-cleavage depletes S in lung but not nasal epithelial cells. At the infected cell membrane during cell-cell spread, TMPRSS2 mediates S FCS cleavage but is negatively regulated in the presence of ACE2. Tissue-specific solute carriers SLC6A19 and SLC6A20 sequester ACE2, thereby relieving inhibition of TMPRSS2 and enabling S FCS cleavage during cell-cell spread. Critically, whilst all SARS-CoV-2 lineages benefit from this latter pathway, Omicron variants have evolved exclusive dependence on it—a strategic consolidation that focuses S proteolytic activation to TMPRSS2 alone. The combined outcomes of S protein regulation across viral and cell membranes reveal how tissue-specific proteolytic optimisation drives Omicron’s transmission fitness advantage in the upper respiratory tract but at the cost of heavy attenuation in the lower respiratory tract. Abstract Figure: Graphical Abstract: Dynamics of Spike pools, S FCS cleavage and ACE2 conformations collectively define Omicron Tropism A & B. Spatial regulation of S protein FCS cleavage. Viral S protein can traffic to two distinct cellular locations: A. virion particles within the trans-Golgi network of infected cells, or B. the plasma membrane of infected cells. Proteolytic cleavage of the FCS at each location is governed by different mechanisms. C. In virion particles, furin plays the dominant role, mediating hyper-FCS cleavage that culminates in S protein depletion in virions generated from cells derived from the lung. Nasal epithelia and other cells permissive to Omicron infections, sustain efficient FCS cleavage without S protein virion depletion. Over time, this phenotype has continued to consolidate from the pandemic to endemic stages of SARS-CoV-2 (Spike depletion relative to Global frequencies of major variants (courtesy of Nextstrain)). The arrival of JN.1 sub-lineages observed the peak of S protein depletion from furin and furin like protease hyper-cleavage. D. From the consequences of S protein depletion, the role of non-FCS cleaved S at the infected cell membrane is revealed. At the infected cell membrane (cells in blue) during cell-cell contact, TMPRSS2 is the primary FCS protease and is present on the recipient cell membrane (cells in green). mportantly, ACE2 negatively regulates TMPRSS2 activity at this location; however, this inhibition is circumvented when ACE2 forms complexes with solute carriers SLC6A19 or SLC6A20. This interaction frees TMPRSS2 from ACE2-mediated inhibition, enabling efficient S1/S2 FCS cleavage at the cell membrane by TMPRSS2. This subsequently exposes S2’ for TMPRSS2 cleavage, driving efficient cell-cell spread via a common protease-gated mechanism. Efficient cell-cell spread is sustained across all SARS-CoV-2 lineages using this pathway, and more recently demonstrated to afford JN.1 sub-lineages a itness advantage relative to XBB.1.5 sub-lineages.

People with HIV have an increased cardiovascular disease (CVD) risk due to both HIV and adverse effects of treatments. The currently preferred class of anti-retroviral drugs, integrase strand transfer inhibitors (INSTIs), has variably been linked to higher CVD risk and weight gain. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) reduce CVD events and heart failure hospitalisations in people with or without type 2 diabetes, while also reducing weight and blood pressure, but have not been studied in people with HIV. Pitavastatin lowers CVD events in those with HIV, although it is not widely available. OPTIMAR is a 2 × 2 factorial, randomised, placebo-controlled, double-blind, phase III/IV trial and will examine the efficacy and safety of dapagliflozin 10 mg versus placebo on metabolic parameters and of rosuvastatin 10 mg + ezetimibe 10 mg versus pitavastatin 4 mg. A cardiac substudy will determine the effect of dapagliflozin versus placebo on epicardial adipose tissue (EAT) volume and coronary plaque characteristics. Virologically suppressed individuals with HIV on INSTI-based ART aged 40-75 years who had a significant weight gain after starting INSTI or BMI ≥ 30 kg/m² will be included. Participants will be randomised 1:1 to blinded dapagliflozin or placebo, followed by 1:1 to open-label rosuvastatin + ezetimibe or pitavastatin for 48 weeks. The primary endpoints will be body weight and LDL reduction for the SGLT2i and statin randomisations respectively in the modified intention-to-treat population. Follow-up will continue to 48 weeks with primary analysis at week-24 and follow-up analysis at week-48. OPTIMAR is the first trial to investigate the use of SGLT2i as a cardiometabolic intervention in people with HIV. Demonstrating favourable cardiometabolic effects of SGLT2i will introduce an innovative approach to primary CVD prevention in this at-risk population and support conducting larger trials evaluating clinical endpoints including major adverse cardiovascular events. If the combination of rosuvastatin and ezetimibe is found to be similar or superior to pitavastatin, this will inform a more feasible lipid management approach for people with HIV internationally, especially in resource-limited settings. The cardiac sub-study will further clarify the cardioprotective role of SGLT2i and provide mechanistic insights. ClinicalTrials.gov, NCT06317051 (https://clinicaltrials.gov/study/NCT06317051?cond=HIV&intr=Dapagliflozin&aggFilters=status:not%20rec&rank=2). Registration on 21 February 2024.

Efficient and secure integration of epidemiological data with pathogen genome sequence data is essential for identification of transmission clusters, monitoring of emerging mutations and targeting public health responses. However, this information is often collected across different organisations: epidemiological data is collected by public health units while genome sequence data is collected by diagnostic laboratories. Linking these sources often requires manual or semi-manual approaches, leading to unnecessary delays in identifying emerging outbreaks. To address this, we developed a proof-of-concept privacy-preserving distributed platform, SecureEpiLink, for automatic linkage of pathogen genome sequences and notification data across public health and diagnostic laboratories. SecureEpiLink uses cryptographic hashing to establish linkage, without exposing personal identifying information. This ensures that the resulting linked data can be used to identify the emergence of transmission clusters without identification of individuals comprising each cluster. The original identifiable data can continue to be stored at source labs. We benchmarked SecureEpiLink against manual linkage and another linkage service using HIV and HCV datasets from New South Wales, Australia. SecureEpiLink performed similarly to manual linkage and outperformed previously used linkage algorithms, with all errors attributable to data entry errors in the underlying dataset. Lastly, we demonstrate how SecureEpiLink can be integrated with automated genomic epidemiology pipelines. SecureEpiLink is available from https://github.com/jolenefarrell/SecureEpiLink . Author summary: Linkage of epidemiological data collected in public health units with pathogen genome sequence data generated by diagnostic labs in real-time is essential for targeted public health responses. However, linkage creates risks of stigmatisation and criminalisation for individuals. We have designed, implemented and tested a privacy preserving real-time system for linking pathogen genome sequence data with public health notification data within different jurisdictions.

Background People with HIV have an increased cardiovascular disease (CVD) risk due to both HIV and adverse effects of treatments. The currently preferred class of anti-retroviral drugs, integrase strand transfer inhibitors (INSTIs), have variably been linked to higher CVD risk and weight gain. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) reduce CVD events and heart failure hospitalisations in people with or without type 2 diabetes, while also reducing weight and blood pressure, but have not been studied in people with HIV. Pitavastatin lowers CVD events in those with HIV, although it is not widely available. Methods OPTIMAR is a 2x2 factorial, randomised, placebo-controlled, double-blind, phase III/IV trial and will examine the efficacy and safety of dapagliflozin 10mg versus placebo on metabolic parameters and of rosuvastatin 10mg + ezetimibe 10mg versus pitavastatin 4mg. A cardiac substudy will determine the effect of dapagliflozin versus placebo on epicardial adipose tissue (EAT) volume and coronary plaque characteristics. Adults aged 40–75 years with HIV with weight gain or obesity who have been virologically stable on INSTI-based ART for over 12 months will be included. Participants will be randomised 1:1 to blinded dapagliflozin or placebo, followed by 1:1 randomisation to open label rosuvastatin + ezetimibe or pitavastatin. The primary endpoints will be body weight and LDL reduction for the SGLT2i and statin randomisations respectively in the modified intention-to-treat population. Follow-up will continue to 48 weeks with primary analysis at week-24 and follow-up analysis at week-48. Discussion OPTIMAR is the first trial to investigate the use of SGLT2i as a cardiometabolic intervention in people with HIV. Demonstrating favourable cardiometabolic effects of SGLT2i will introduce an innovative approach to primary CVD prevention in this at-risk population and support conducting larger trials evaluating clinical endpoints including major adverse cardiovascular events. If the combination of rosuvastatin and ezetimibe is found to be similar or superior to pitavastatin, this will inform a more feasible lipid management approach for people with HIV internationally, especially in resource-limited settings. The cardiac sub-study will further clarify the cardioprotective role of SGLT2i and provide mechanistic insights. Trial registration: ClinicalTrials.gov, NCT06317051 (https://clinicaltrials.gov/study/NCT06317051?cond=HIV&intr=Dapagliflozin&aggFilters=status:not%20rec&rank=2). Registration date: 21 February 2024.

Background: D²EFT (Dolutegravir and Darunavir Evaluation in Adults Failing Therapy) was a phase 3b/4 randomized clinical trial designed to assess second-line treatment options systematically. This substudy evaluated the distribution of drug resistance mutations (DRMs) before treatment randomization in individuals failing first-line therapy. Methods: From a total of 826 participants across 14 countries, 727 sequences that covered the PR-RT-INT (700), PR-RT (24), and RT (3) regions were analyzed for drug resistance. Sequences were submitted to the Stanford HIV drug resistance database to detect DRMs and assign subtypes. By adjusting for country and antiretroviral therapy regimen, we assessed the association between DRMs and country and reported DRMs. Results: Subtype C of human immunodeficiency virus type 1 (HIV-1) accounted for most (59.3%) infections. There were extraordinarily high rates of high-level resistance to lamivudine and emtricitabine (both at 88.3%) and for efavirenz and nevirapine (92.8% and 96.8%, respectively). On average, nucleoside reverse transcriptase inhibitor mutations had the highest occurrence across countries with M184V/I detected in 86.2% of the samples, while the highest proportion of nonnucleoside reverse transcriptase inhibitor mutations was K103N at 57.8%. K103N had an increased likelihood of occurrence in African and South American countries (P < .05). Participants with prior exposure to a zidovudine-containing regimen had an increased likelihood of T215F/Y mutations (6.80 [2.59–17.86]), while those with nevirapine/rilpivirine exposure had a decreased likelihood of K103N mutations (0.29 [0.15–0.56]). Conclusions: The regional specificity of mutations underscores the dynamic nature of HIV-1 drug resistance patterns and the importance of monitoring and understanding local mutation profiles.

COVID-19 has resulted in over 777 million confirmed cases and more than 7 million deaths globally. While vaccination offers protection for individuals with a functional immune system, immunocompromised populations will not generate sufficient responses, highlighting the critical need for new antiviral treatments. Here we evaluated four highly conserved anti-COVID siRNAs targeting the ORF1a-Nsp1, Membrane, and Nucleocapsid regions by identifying their antiviral efficacy in vitro and investigated the direct delivery of naked siRNAs to the respiratory tract of mice via intranasal instillation to provide proof-of-concept evidence of their in vivo antiviral activity. Dose-response analysis of siRNAs revealed a range of IC50 0.02 nM to 0.9 nM. Intranasal administration of naked anti-COVID siRNA-18 in a K18-hACE2 transgenic SARS-CoV-2 mouse model was capable of reducing viral mRNA levels and disease severity. While anti-COVID siRNA-30 induced modest interferon-stimulated gene expression in vitro and immune cell infiltration in vivo, these effects were markedly reduced by 2′-O-methyl-AS456 chemical modification, which preserved antiviral efficacy against SARS-CoV-2 while minimizing off-target immune activation. These results demonstrate the feasibility of direct respiratory siRNA administration for in vivo viral suppression and highlight the benefit of using conserved target sequences and chemical modification to enhance therapeutic safety and efficacy.