Viral and host mediators of non-suppressible HIV-1 viremia.

Non-suppressible HIV-1 viremia (NSV) is defined as persistent low-level viremia on antiretroviral therapy (ART) without evidence of ART non-adherence or significant drug resistance. Unraveling the mechanisms behind NSV would broaden our understanding of HIV-1 persistence. Here we analyzed plasma virus sequences in eight ART-treated individuals with NSV (88% male) and show that they are composed of large clones without evidence of viral evolution over time in those with longitudinal samples. We defined proviruses that match plasma HIV-1 RNA sequences as 'producer proviruses', and those that did not as 'non-producer proviruses'. Non-suppressible viremia arose from expanded clones of producer proviruses that were significantly larger than the genome-intact proviral reservoir of ART-suppressed individuals. Integration sites of producer proviruses were enriched in proximity to the activating H3K36me3 epigenetic mark. CD4+ T cells from participants with NSV demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, participants with NSV showed significantly lower HIV-specific CD8+ T cell responses compared with untreated viremic controllers with similar viral loads. We identified potential critical host and viral mediators of NSV that may represent targets to disrupt HIV-1 persistence.

T cell responses to SARS-CoV-2 infection and vaccination are elevated in B cell deficiency and reduce risk of severe COVID-19.

Individuals with primary and pharmacologic B cell deficiencies have high rates of severe disease and mortality from coronavirus disease 2019 (COVID-19), but the immune responses and clinical outcomes after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination have yet to be fully defined. Here, we evaluate the cellular immune responses after both SARS-CoV-2 infection and vaccination in patients receiving the anti-CD20 therapy rituximab (RTX) and those with low B cell counts due to common variable immune deficiency (CVID) disease. Assessment of effector and memory CD4+ and CD8+ T cell responses to SARS-CoV-2 revealed elevated reactivity and proliferative capacity after both infection and vaccination in B cell-deficient individuals, particularly within the CD8+ T cell compartment, in comparison with healthy controls. Evaluation of clinical outcomes demonstrates that vaccination of RTX-treated individuals was associated with about 4.8-fold reduced odds of moderate or severe COVID-19 in the absence of vaccine-induced antibodies. Analysis of T cell differentiation demonstrates that RTX administration increases the relative frequency of naïve CD8+ T cells, potentially by depletion of CD8+CD20dim T cells, which are primarily of an effector memory or terminal effector memory (TEMRA) phenotype. However, this also leads to a reduction in preexisting antiviral T cell immunity. Collectively, these data indicate that individuals with B cell deficiencies have enhanced T cell immunity after both SARS-CoV-2 infection and vaccination that potentially accounts for reduced hospitalization and severe disease from subsequent SARS-CoV-2 infection.

Viral and Host Mediators of Non-Suppressible HIV-1 Viremia.

Non-suppressible HIV-1 viremia (NSV) can occur in persons with HIV despite adherence to combination antiretroviral therapy (ART) and in the absence of significant drug resistance. Here, we show that plasma NSV sequences are comprised primarily of large clones without evidence of viral evolution over time. We defined proviruses that contribute to plasma viremia as "producer", and those that did not as "non-producer". Compared to ART-suppressed individuals, NSV participants had a significantly larger producer reservoir. Producer proviruses were enriched in chromosome 19 and in proximity to the activating H3K36me3 epigenetic mark. CD4+ cells from NSV participants demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, NSV participants showed no elevation in HIV-specific CD8+ cell responses and producer proviruses were enriched for HLA escape mutations. We identified critical host and viral mediators of NSV that represent potential targets to disrupt HIV persistence and promote viral silencing.

Screening and characterization of vaginal fluid donations for vaginal microbiota transplantation.

Bacterial vaginosis (BV), the overgrowth of diverse anaerobic bacteria in the vagina, is the most common cause of vaginal symptoms worldwide. BV frequently recurs after antibiotic therapy, and the best probiotic treatments only result in transient changes from BV-associated states to "optimal" communities dominated by a single species of Lactobacillus. Therefore, additional treatment strategies are needed to durably alter vaginal microbiota composition for patients with BV. Vaginal microbiota transplantation (VMT), the transfer of vaginal fluid from a healthy person with an optimal vaginal microbiota to a recipient with BV, has been proposed as one such alternative. However, VMT carries potential risks, necessitating strict safety precautions. Here, we present an FDA-approved donor screening protocol and detailed methodology for donation collection, storage, screening, and analysis of VMT material. We find that Lactobacillus viability is maintained for over six months in donated material stored at - 80 °C without glycerol or other cryoprotectants. We further show that species-specific quantitative PCR for L. crispatus and L. iners can be used as a rapid initial screening strategy to identify potential donors with optimal vaginal microbiomes. Together, this work lays the foundation for designing safe, reproducible trials of VMT as a treatment for BV.

Profound Treg perturbations correlate with COVID-19 severity.

The hallmark of severe COVID-19 is an uncontrolled inflammatory response, resulting from poorly understood immunological dysfunction. We hypothesized that perturbations in FoxP3+ T regulatory cells (Treg), key enforcers of immune homeostasis, contribute to COVID-19 pathology. Cytometric and transcriptomic profiling revealed a distinct Treg phenotype in severe COVID-19 patients, with an increase in Treg proportions and intracellular levels of the lineage-defining transcription factor FoxP3, correlating with poor outcomes. These Tregs showed a distinct transcriptional signature, with overexpression of several suppressive effectors, but also proinflammatory molecules like interleukin (IL)-32, and a striking similarity to tumor-infiltrating Tregs that suppress antitumor responses. Most marked during acute severe disease, these traits persisted somewhat in convalescent patients. A screen for candidate agents revealed that IL-6 and IL-18 may individually contribute different facets of these COVID-19-linked perturbations. These results suggest that Tregs may play nefarious roles in COVID-19, by suppressing antiviral T cell responses during the severe phase of the disease, and by a direct proinflammatory role.

Uncertainty modelling in deep learning for safer neuroimage enhancement: Demonstration in diffusion MRI.

Deep learning (DL) has shown great potential in medical image enhancement problems, such as super-resolution or image synthesis. However, to date, most existing approaches are based on deterministic models, neglecting the presence of different sources of uncertainty in such problems. Here we introduce methods to characterise different components of uncertainty, and demonstrate the ideas using diffusion MRI super-resolution. Specifically, we propose to account for intrinsic uncertainty through a heteroscedastic noise model and for parameter uncertainty through approximate Bayesian inference, and integrate the two to quantify predictive uncertainty over the output image. Moreover, we introduce a method to propagate the predictive uncertainty on a multi-channelled image to derived scalar parameters, and separately quantify the effects of intrinsic and parameter uncertainty therein. The methods are evaluated for super-resolution of two different signal representations of diffusion MR images-Diffusion Tensor images and Mean Apparent Propagator MRI-and their derived quantities such as mean diffusivity and fractional anisotropy, on multiple datasets of both healthy and pathological human brains. Results highlight three key potential benefits of modelling uncertainty for improving the safety of DL-based image enhancement systems. Firstly, modelling uncertainty improves the predictive performance even when test data departs from training data ("out-of-distribution" datasets). Secondly, the predictive uncertainty highly correlates with reconstruction errors, and is therefore capable of detecting predictive "failures". Results on both healthy subjects and patients with brain glioma or multiple sclerosis demonstrate that such an uncertainty measure enables subject-specific and voxel-wise risk assessment of the super-resolved images that can be accounted for in subsequent analysis. Thirdly, we show that the method for decomposing predictive uncertainty into its independent sources provides high-level "explanations" for the model performance by separately quantifying how much uncertainty arises from the inherent difficulty of the task or the limited training examples. The introduced concepts of uncertainty modelling extend naturally to many other imaging modalities and data enhancement applications.

Profound Treg perturbations correlate with COVID-19 severity.

The hallmark of severe COVID-19 disease has been an uncontrolled inflammatory response, resulting from poorly understood immunological dysfunction. We explored the hypothesis that perturbations in FoxP3+ T regulatory cells (Treg), key enforcers of immune homeostasis, contribute to COVID-19 pathology. Cytometric and transcriptomic profiling revealed a distinct Treg phenotype in severe COVID-19 patients, with an increase in both Treg proportions and intracellular levels of the lineage-defining transcription factor FoxP3, which correlated with poor outcomes. Accordingly, these Tregs over-expressed a range of suppressive effectors, but also pro-inflammatory molecules like IL32. Most strikingly, they acquired similarity to tumor-infiltrating Tregs, known to suppress local anti-tumor responses. These traits were most marked in acute patients with severe disease, but persisted somewhat in convalescent patients. These results suggest that Tregs may play nefarious roles in COVID-19, via suppressing anti-viral T cell responses during the severe phase of the disease, and/or via a direct pro-inflammatory role.

SARS-CoV-2 viral load is associated with increased disease severity and mortality.

The relationship between SARS-CoV-2 viral load and risk of disease progression remains largely undefined in coronavirus disease 2019 (COVID-19). Here, we quantify SARS-CoV-2 viral load from participants with a diverse range of COVID-19 disease severity, including those requiring hospitalization, outpatients with mild disease, and individuals with resolved infection. We detected SARS-CoV-2 plasma RNA in 27% of hospitalized participants, and 13% of outpatients diagnosed with COVID-19. Amongst the participants hospitalized with COVID-19, we report that a higher prevalence of detectable SARS-CoV-2 plasma viral load is associated with worse respiratory disease severity, lower absolute lymphocyte counts, and increased markers of inflammation, including C-reactive protein and IL-6. SARS-CoV-2 viral loads, especially plasma viremia, are associated with increased risk of mortality. Our data show that SARS-CoV-2 viral loads may aid in the risk stratification of patients with COVID-19, and therefore its role in disease pathogenesis should be further explored.

Structural topology defines protective CD8+ T cell epitopes in the HIV proteome.

Mutationally constrained epitopes of variable pathogens represent promising targets for vaccine design but are not reliably identified by sequence conservation. In this study, we employed structure-based network analysis, which applies network theory to HIV protein structure data to quantitate the topological importance of individual amino acid residues. Mutation of residues at important network positions disproportionately impaired viral replication and occurred with high frequency in epitopes presented by protective human leukocyte antigen (HLA) class I alleles. Moreover, CD8+ T cell targeting of highly networked epitopes distinguished individuals who naturally control HIV, even in the absence of protective HLA alleles. This approach thereby provides a mechanistic basis for immune control and a means to identify CD8+ T cell epitopes of topological importance for rational immunogen design, including a T cell-based HIV vaccine.