Abha Chopra

We report the complete mitochondrial genome of the halotolerant green alga Dunaliella salina CS-265, isolated from a hypersaline lake in central Australia. The genome is a circular DNA molecule of 30,073 bp, encoding seven protein-coding genes, nine rRNAs, and three tRNAs. Four core genes (cox1, cob, nad1, and nad5) are fragmented by multiple introns, whereas others remain intact. The absence of ATP synthase subunits and ribosomal protein genes reflects ongoing reductive evolution in Dunaliella mitochondria. This genome adds a new organellar resource from an Australian isolate, complementing previous studies and providing further insight into mitochondrial genome dynamics in halotolerant green algae.

Mutations in human immunodeficiency virus type 1 (HIV-1) enable the virus to evade recognition and killing by human leucocyte antigen (HLA)-restricted T cells. These viral adaptations are specific to the HLA type of individuals and are therefore evident as HLA allele-HIV sequence associations at the population level. Most studies of HLA associations have been cross-sectional and may not capture selective changes that have accumulated to reach fixation at the population level, with potential impacts on viral replication and clinical outcomes. In this study, we examined the population from Western Australia, where HLA-HIV-1 associations were first demonstrated, to determine if ongoing evolution has occurred over more than 30 years of observation.
Cross-sectional HIV-1 subtype B sequences sampled at two time points, early in the epidemic (1992 - 2002, n = 182) and recently (2017 - 2022, n = 119) was utilised to examine HIV-1 evolutionary dynamics overtime. In addition, HIV-1 subtype B viral load records (one measurement per individual) from a five-year period early in the epidemic (1997 - 2002, n = 673) were compared with recent data (2017 - 2022, n = 363) to determine whether any population level HIV-1 adaptation has functional impact.
The analysis identified 120 amino acid positions across the Gag, Pol and Nef genes that showed significant change in proportion over time, with most (100/120; 83.3%) showing an increase in the proportion of one or more of the non-consensus amino acids. Of these positions, 35% (42/120) included one or more amino acids (48; 34 in Pol, 9 in Gag and 5 in Nef) reported as HLA-associated viral adaptations (35/48; 72.9%) or putative compensatory adaptations (11/48, 22.9%). Over two thirds of these adaptations (68.8%; 33/48) increased in proportion over time (range 5.8% to 46%), with eight becoming the consensus sequence. We also observed the accumulation of specific compensatory mutations within epitopes presented by protective HLA alleles. Other accumulated non-consensus amino acid changes (38/120) were predicted to weaken the peptide-HLA binding affinity of known HIV T cell epitopes, suggesting that the previously published list of HLA-associated viral adaptations used in our study was not exhaustive. Only two Pol reverse transcriptase non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations showed a significant change in proportion over time (K256Q [K101Q in reverse transcriptase region]; 22.9%, P-adjusted <0.001 and K258N [K103N in reverse transcriptase region]; 7.7%, P-adjusted = 0.020). Notably, we highlight the significant accumulation of adaptations (Gag: R76K, 40.8%, P-adjusted <0.001; H219Q, 25.8%, P-adjusted = 0.020 and R286K, 24.4%, P-adjusted = 0.036) that confer adaptation to both HLA-restricted T cell immune responses and antiretroviral therapy. There was a significant increase in baseline viral load between the two time periods examined (P ⟨0.001, OR = 2.4).
These findings provide evidence of ongoing HIV-1 adaptation to human immune responses at the population level, with a likely increase in virulence, as captured by viral load. The enrichment of viral adaptations within circulating strains may lead to loss of immune targets for prevalent immune responses and has important implications for vaccine development and cure strategies.

The abundant skin commensal, Staphylococcus epidermidis, is the leading cause of late-onset sepsis (LOS) in preterm infants but rarely causes infections in term infants and adults. Staphylococcal virulence mechanisms and the role of the preterm immune responses in driving these life-threatening infections remain poorly understood. Using an ex vivo sepsis model, we challenged whole blood from very preterm infants (30-32 weeks gestational age, GA; n = 8), term infants (> 37 weeks GA; n = 8), and young adults (18-25 years; n = 8) with either live S. epidermidis or S. aureus (~ 10
colony-forming units, CFU/ml) for 90 min. Dual RNA-sequencing (RNA-seq) was performed to simultaneously assess host and pathogen gene expression profiles, identifying common and pathogen-specific responses across cohorts. We found shared immune processes induced in all age groups upon bacterial challenge, including cytokine (IL1A, IL1B, IL6, IFNB1) and chemokine (CCL20, CCL3, CCL7, CXCL2) signalling. Preterm infants also exhibited unique responses, such as increased platelet activation and fibrin clot formation, Wnt signalling, and hypoxia pathways in response to S. epidermidis challenge. Our findings suggest that bacterial gene co-expression, including iron acquisition and heme biosynthesis genes, are also influenced by the hosts developmental age, highlighting the complexity of host-bacterial interactions in the early stages of neonatal sepsis.

Purpose: Despite ART initiation in the acute phase of HIV infection (AHI), the viral reservoir persists. In the NOVA study, the viral reservoir declined between 24 and 156 weeks after ART initiation in individuals treated during AHI and correlated with the presence of HIV-specific CD8+ T-cell responses at 24 weeks. Now we further explore the effect of selective CD8+ T-cell pressure on viral integration sites.

Method: Two individuals participating in the Netherlands Cohort Study on Acute HIV Infection (NOVA study) (P1 and P2) were selected for an in-depth study (clinical characteristics shown in Fig. 1). HIV-specific CD8+ T-cell proliferative responses upon HIV peptide stimulation were determined by flow cytometry. Viral isolates were sequenced and analyzed for the presence of CD8+ T-cell epitopes using a HLA-I class binding prediction tool. HIV proviral structure and integration sites were characterized using a modified Integrated Proviral Sequencing Assay (IPSA).
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Results: In P1, strong, broad HIV-specific proliferative CD8+ T-cell responses were observed at 24 weeks, which corresponded with a substantial number of viral escape mutations in HLA-A*02:01:01, HLA-B*13:02:01, HLA-B*15:01:01 (Gag), HLA-B*13:02:01 and HLA-B*15:01:01 (Nef) restricted epitopes. In P2, low frequencies of proliferating CD8+ T-cells were observed at 24 weeks, which is reflected by a relatively low number of escape mutations at that time point. In P2 viral escape was mostly observed in HLA-B*40:01:02 (Gag) restricted epitopes and in HLA-A*01:01 and HLA-A*02:01:01 (Nef and Pol) restricted epitopes (Fig. 2). IPSA demonstrated a strong decrease in intact HIV DNA between 24 and 156 weeks in both participants. Interestingly, 65-77% of intact proviruses were located in a genic site, of which 33-66% in antisense region implying selection for transcriptionally latent proviruses.
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Conclusions: This study found that broad HIV-specific CD8+ T-cell responses drive viral escape mutations and associates with proviral loss and propensity of antisense (silent) integration.

Co-trimoxazole is a leading global cause of severe cutaneous adverse drug reactions (SCAR) including Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS). Co-trimoxazole-induced SCAR are associated with HLA class I alleles including HLA-B*13:01 and HLA-B*38:02 in Southeast Asian (SEA) populations. However, the global generalizability of these associations is unknown but critical for population-appropriate risk stratification and diagnosis.
To determine HLA risk factors associated with co-trimoxazole-induced SJS/TEN and DRESS in populations from the United States (US) and South Africa (SA).
We performed high-resolution HLA typing on dermatologist-adjudicated co-trimoxazole-induced SCAR patients in the US (n=63) and SA (n=26) compared to population controls. Peptide binding and docking analyses were performed using MHCcluster2.0 and CB-Dock2.
In a multiple logistic regression model, HLA-B*44:03 (Pc<0.001, OR: 4.08), HLA-B*38:01 (Pc<0.001, OR: 5.66), and HLA-C*04:01 (Pc=0.003, OR: 2.50) were independently associated with co-trimoxazole-induced SJS/TEN in the US. HLA-B*44:03 was also associated with co-trimoxazole-induced DRESS in SA (Pc=0.019, OR: 10.69). Distinct HLA-B variants with shared peptide binding specificities (SPBS) and HLA-C*04:01 identified 94% and 78% of co-trimoxazole-induced SJS/TEN and DRESS in the US, respectively. The SEA risk allele HLA-B*13:01, with SPBS to HLA-B*44:03, was identified in just 1/63 US SCAR patients.
HLA alleles with SPBS to SEA-related risk alleles including HLA-B*44:03 (SPBS with HLA-B*13:01) and HLA-B*38:01 (SPBS with HLA-B*38:02) but also HLA-C*04:01 predisposed to co-trimoxazole-induced SCAR in the US and SA. These findings provide biological plausibility and strategies for global risk prediction and diagnosis of co-trimoxazole-induced SCAR.
HLA alleles including HLA-B*13:01 and HLA-B*38:02 are risk factors for co-trimoxazole-induced SCAR in Asian populations. However, the generalizability of these associations to other global populations is unknown but critical for population-appropriate risk stratification and diagnosis.
HLA alleles with shared peptide binding specificities (SPBS) to Asian-related risk alleles including HLA-B*44:03 (SPBS with HLA-B*13:01) and HLA-B*38:01 (SPBS with HLA-B*38:02) but also HLA-C*04:01 predisposed to co-trimoxazole-induced SCAR in the US and South Africa.
HLA alleles previously associated with co-trimoxazole-induced SCAR do not identify risk across populations. However, HLA alleles with SPBS provide biological plausibility and strategies for global and population-appropriate clinical risk stratification and diagnosis of cotrimoxazole-induced SCAR.

Few HIV-specific epitopes restricted by non-classical HLA-E have been described, and even less is known about the functional profile of responding CD8 T cells (CD8s). This study evaluates the functional characteristics of CD8s targeting the Gag epitope KF11 (KAFSPEVIPMF) restricted by either HLA-E (E-CD8s) or HLA-B57 (B57-CD8s). CD8s from eight people with HIV (PWH) were cocultured with KF11 peptide presented by cell lines expressing HLA-B*57:01, HLA-E*01:01 or E*01:03. CD8 responses were analyzed using scRNA-seq and scTCR-seq. Supernatants were also assessed for soluble protein profiling. HLA-I multimers were developed to identify CD8s restricted by HLA-B57 and/or HLA-E ex vivo. B57-CD8s secreted higher levels of cytotoxic cytokines such as IFNγ, whereas E-CD8s produced more chemotactic cytokines, including RANTES, CXCL10 (IP-10), and IL27, findings which were corroborated through scRNA sequencing. TCR clonotypes stimulated by KF11 were cross-restricted by HLA-B*57 and HLA-E*01/03 as demonstrated by in vitro T cell reporter assays and ex vivo multimer screening. Ex vivo CD8s were singly restricted by HLA-B57 and HLA-E, with dual restriction only observed in PWH with lower viral load. These findings demonstrate that certain HIV-specific CD8s in PWH exhibit dual restriction by HLA-B*57 and HLA-E*01/03, leading to functionally distinct immune responses depending upon the restricting allele(s).

Background: Co-trimoxazole (Co-T), an antibiotic used globally for bacterial infections and opportunistic infection prophylaxis is one of the most common causes of Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN); a life-threatening CD8+ T cell-dependent HLA-class I-restricted blistering drug eruption. We aimed to elucidate the differential single-cell immunopathology of Co-T SJS/TEN in persons living with HIV (PLWH) and without HIV (PLWoH).

Method: We obtained blister fluid cells (BC) from three populations: 1) Co-T SJS/TEN PLWH, all of whom were co-infected with tuberculosis (TB) (n = 5); PLWoH (n = 6), or 3) burns patients as control (n = 6). BC were analysed by 5’ scRNA-TCR-CITE-seq with bioinformatic normalization (CellRanger v9.0), transcriptome-based clustering (Seurat v5), and TCRαβ (ClusTCR), differential (VGAS), and proportional analyses (scCODA). Affected skin from two PLWoH with paired BC were analysed by 10x Visium-HD spatial transcriptomics.

Results: Private oligoclonal TCRαβ in blister fluid across Co-T SJS/TEN patients with shared HLA-class I peptide binding specificities were expressed by the same two populations of cytotoxic (GNLY, GZMB, PRF1) and proliferative (STMN1, TUBA1B, H2AFZ) CD8+ T-cells enriched in SJS/TEN and in contact with spatially resolved keratinocytes at the epidermal junction. At late timepoints pathogenic CD8+ T-cells were reduced while M1-like (STAT1, HLA) phagocytes transitioned to M2-like (RNASE1, LIPA) signatures indicative of wound repair. In Co-T SJS/TEN PLWH compared to PLWoH, CD8+ T-cells expressed markers of inactivation (EEF1A1, BTG1, ZFP36L2) and reduced expression of cytotoxic mediators (GNLY, GZMB). PLWH were further discerned by CD1C dendritic cells differentially enriched for markers of activation (CD83, CALR, AIF1), monocytes and macrophages for markers of immune regulation (THBS1, TFGBI, VCAN, STAB1), and CD4+ T-cells for markers of cytotoxicity (GNLY, GZMA), phosphodiesterase-signalling (PDE3B, PDE7B), and immune dysregulation (TFGB1, CRIP1).

Conclusion: We identify putative pathogenic TCR to model shared drug- and HLA-restriction and cell signatures that discern early (cytotoxic) from late (repair) disease processes and a unique cellular sub phenotype of SJS/TEN in PLWH associated with immune dysregulation. These data provide avenues toward personalised treatment approaches for patients with earlier- or later-stage disease and sub phenotypes of SJS/TEN associated with infectious co-morbidities.

The success of cancer immunotherapies has highlighted the importance of monitoring the anti-tumour T cell response. Patients with mesothelioma frequently present with a malignant pleural effusion (MPE) that is commonly drained regularly to alleviate symptoms. As MPE contains tumour cells, T cells and cytokines, it provides a unique opportunity to sample immune events at the tumour site. However, there is minimal information on how MPE T cells are distinct from those in the blood, and whether T cell phenotypes unique to each compartment correlate with survival. We characterised T cell populations of matched MPE and blood from 31 mesothelioma patients using flow cytometry and bulk T cell receptor beta (TCRβ) sequencing. MPE CD8+ and CD4+ T cells displayed increased expression of PD-1, TIGIT, LAG-3 and TIM-3 compared to blood, with co-expression of inhibitory receptors greatest on MPE CD8+ T cells with a tissue resident memory T cell phenotype (CD69+CD103+). CD8+ TCRβ repertoires displayed clonal overlap between MPE and blood, suggesting that a majority of T cells traffic between these compartments. Finally, we show that high expression of PD-1 on circulating CD4+ T cells is an independent prognostic factor for poor survival in this patient group. This work suggests that MPE T cell phenotypes differ from those in circulation, with blood-based T cell subsets more sensitive predictors of outcome in this study.

Inclusion body myositis (IBM) is a late-onset, treatment-resistant inflammatory myopathy. Approximately half of IBM patients develop autoantibodies against cytosolic 5′-nucleotidase 1A (cN1A), but their role in disease pathogenesis remains unclear. This pilot study examined the effects of anti-cN1A-positive IBM serum on human primary myotubes’ transcriptome profile, using anti-cN1A-negative IBM and healthy sera as controls. Exposure to anti-cN1A-positive serum altered the expression of 1126 genes, with upregulation of adaptive immune response genes, notably CTSH and CTSZ, encoding cathepsins H and Z. These findings were validated using a publicly available independent dataset comprising transcriptomes from fresh muscle tissue samples. NT5C1A mRNA, which encodes cN1A, was not detected in cultured myotubes regardless of the presence of autoantibodies. The findings suggest distinct pathological mechanisms in anti-cN1A-positive IBM, independent of direct antibody-target interactions. The role of cathepsins in IBM pathogenesis warrants further investigation.

Background
Co-trimoxazole is a leading global cause of severe cutaneous adverse drug reactions (SCAR) including Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS). Co-trimoxazole-induced SCAR are associated with HLA class I alleles including HLA-B*13:01 and HLA-B*38:02 in Southeast Asian (SEA) populations. However, the global generalizability of these associations is unknown but critical for population-appropriate risk stratification and diagnosis.

Objective
To determine HLA risk factors associated with co-trimoxazole-induced SJS/TEN and DRESS in populations from the United States (US) and South Africa (SA).

Methods
We performed high-resolution HLA typing on dermatologist-adjudicated co-trimoxazole-induced SCAR patients in the US (n=63) and SA (n=26) compared to population controls. Peptide binding and docking analyses were performed using MHCcluster2.0 and CB-Dock2.

Results
In a multiple logistic regression model, HLA-B*44:03 (Pc<0.001, OR: 4.08), HLA-B*38:01 (Pc<0.001, OR: 5.66), and HLA-C*04:01 (Pc=0.003, OR: 2.50) were independently associated with co-trimoxazole-induced SJS/TEN in the US. HLA-B* 44:03 was also associated with co-trimoxazole-induced DRESS in SA (Pc=0.019, OR: 10.69). Distinct HLA-B variants with shared peptide binding specificities (SPBS) and HLA-C*04:01 identified 94% and 78% of co-trimoxazole-induced SJS/TEN and DRESS in the US, respectively. The SEA risk allele HLA-B*13:01, with SPBS to HLA-B*44:03, was identified in just 1/63 US SCAR patient.

Conclusion
HLA alleles with SPBS to SEA-related risk alleles including HLA-B*44:03 (SPBS with HLA-B*13:01) and HLA-B*38:01 (SPBS with HLA-B*38:02) but also HLA-C*04:01 predisposed to co-trimoxazole-induced SCAR in the US and SA. These findings provide biological plausibility and strategies for global risk prediction and diagnosis of co-trimoxazole-induced SCAR.