Metasurfaces are engineered nanostructured interfaces that extend the photonic behavior of natural materials, and they spur many breakthroughs in multiple fields, including quantum optics, ...optoelectronics, and biosensing. Recent advances in metasurface nanofabrication enable precise manipulation of light–matter interactions at subwavelength scales. However, current fabrication methods are costly and time‐consuming and have a small active area with low reproducibility due to limitations in lithography, where sensing nanosized rare biotargets requires a wide active surface area for efficient binding and detection. Here, a plastic‐templated tunable metasurface with a large active area and periodic metal–dielectric layers to excite plasmonic Fano resonance transitions providing multimodal and multiplex sensing of small biotargets, such as proteins and viruses, is introduced. The tunable Fano resonance feature of the metasurface is enabled via chemical etching steps to manage nanoperiodicity of the plastic template decorated with plasmonic layers and surrounding dielectric medium. This metasurface integrated with microfluidics further enhances the light–matter interactions over a wide sensing area, extending data collection from 3D to 4D by tracking real‐time biomolecular binding events. Overall, this work resolves cost‐ and complexity‐related large‐scale fabrication challenges and improves multilayer sensitivity of detection in biosensing applications.
Metasurface fabrication techniques are costly and time‐consuming since multiple complex deposition and patterning steps are required to build a device with a small active area. The digital versatile disk (DVD)‐templated metasurface approach allows the fabrication of a cost‐effective Fano resonant metasurface over a large active area that provides layer‐by‐layer biosensing of binding and interactions of protein, antibody, and virus particles.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
We describe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses, soluble markers of inflammation, and antibody levels and neutralization capacity longitudinally in ...70 individuals with PCR-confirmed SARS-CoV-2 infection. Participants represent a spectrum of illness and recovery, including some with persistent viral shedding in saliva and many experiencing post-acute sequelae of SARS-CoV-2 infection (PASC). T cell responses remain stable for up to 9 months. Whereas the magnitude of early CD4+ T cell immune responses correlates with severity of initial infection, pre-existing lung disease is independently associated with higher long-term SARS-CoV-2-specific CD8+ T cell responses. Among participants with PASC 4 months following coronavirus disease 2019 (COVID-19) symptom onset, we observe a lower frequency of CD8+ T cells expressing CD107a, a marker of degranulation, in response to Nucleocapsid (N) peptide pool stimulation, and a more rapid decline in the frequency of N-specific interferon-γ-producing CD8+ T cells. Neutralizing antibody levels strongly correlate with SARS-CoV-2-specific CD4+ T cell responses.
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•The magnitude of early CD4+ T cell responses correlates with severity of COVID-19•Prior lung disease correlates with higher SARS-CoV-2-specific CD8+ T cell responses•PASC is associated with a decline in N-specific interferon-γ-producing CD8+ T cells•Neutralizing capacity correlates with SARS-CoV-2-specific CD4+ T cell responses
CD4+ and CD8+ T cell responses following natural infection with COVID-19 are stable over 8 months. Individuals with PASC demonstrate a lower frequency of CD8+ T cells expressing CD107a, a marker of degranulation, and a more rapid decline in the frequency of N-specific interferon-γ-producing CD8+ T cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ...ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
A major obstacle to achieving long-term antiretroviral (ART) free remission or functional cure of HIV infection is the presence of persistently infected cells that establish a long-lived ...viral reservoir. HIV largely resides in anatomical regions that are inaccessible to routine sampling, however, and non-invasive methods to understand the longitudinal tissue-wide burden of HIV persistence are urgently needed. Positron emission tomography (PET) imaging is a promising strategy to identify and characterize the tissue-wide burden of HIV. Here, we assess the efficacy of using immunoPET imaging to characterize HIV reservoirs and identify anatomical foci of persistent viral transcriptional activity using a radiolabeled HIV Env-specific broadly neutralizing antibody,
89
Zr-VRC01, in HIV-infected individuals with detectable viremia and on suppressive ART compared to uninfected controls (NCT03729752). We also assess the relationship between PET tracer uptake in tissues and timing of ART initiation and direct HIV protein expression in CD4 T cells obtained from lymph node biopsies. We observe significant increases in
89
Zr-VRC01 uptake in various tissues (including lymph nodes and gut) in HIV-infected individuals with detectable viremia (
N
= 5) and on suppressive ART (
N
= 5) compared to uninfected controls (
N
= 5). Importantly, PET tracer uptake in inguinal lymph nodes in viremic and ART-suppressed participants significantly and positively correlates with HIV protein expression measured directly in tissue. Our strategy may allow non-invasive longitudinal characterization of residual HIV infection and lays the framework for the development of immunoPET imaging in a variety of other infectious diseases.
The major barrier to an HIV cure is the HIV reservoir: latently-infected cells that persist despite effective antiretroviral therapy (ART). There have been few cohort-based studies evaluating host ...genomic or transcriptomic predictors of the HIV reservoir. We performed host RNA sequencing and HIV reservoir quantification (total DNA tDNA, unspliced RNA usRNA, intact DNA) from peripheral CD4+ T cells from 191 ART-suppressed people with HIV (PWH). After adjusting for nadir CD4+ count, timing of ART initiation, and genetic ancestry, we identified two host genes for which higher expression was significantly associated with smaller total DNA viral reservoir size, P3H3 and NBL1, both known tumor suppressor genes. We then identified 17 host genes for which lower expression was associated with higher residual transcription (HIV usRNA). These included novel associations with membrane channel (KCNJ2, GJB2), inflammasome (IL1A, CSF3, TNFAIP5, TNFAIP6, TNFAIP9, CXCL3, CXCL10), and innate immunity (TLR7) genes (FDR-adjusted q<0.05). Gene set enrichment analyses further identified significant associations of HIV usRNA with TLR4/microbial translocation (q = 0.006), IL-1/NRLP3 inflammasome (q = 0.008), and IL-10 (q = 0.037) signaling. Protein validation assays using ELISA and multiplex cytokine assays supported these observed inverse host gene correlations, with P3H3, IL-10, and TNF-α protein associations achieving statistical significance (p<0.05). Plasma IL-10 was also significantly inversely associated with HIV DNA (p = 0.016). HIV intact DNA was not associated with differential host gene expression, although this may have been due to a large number of undetectable values in our study. To our knowledge, this is the largest host transcriptomic study of the HIV reservoir. Our findings suggest that host gene expression may vary in response to the transcriptionally active reservoir and that changes in cellular proliferation genes may influence the size of the HIV reservoir. These findings add important data to the limited host genetic HIV reservoir studies to date.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Elite controllers (EC), a small subset of the HIV-positive population (< 1%), suppress HIV viremia below the limit of quantification of clinical viral load assays in the absence of antiretroviral ...therapy (ART). However, there is a paucity of longitudinal data detailing the viral and immune dynamics or HIV reservoir seeding during acute infection in individuals that go on to become Elite Controllers. This case provides important information regarding the establishment of elite HIV control during acute infection and also demonstrates an increase in HIV-specific immune responses following ART despite undetectable peripheral blood cellular measures of HIV persistence. This case also highlights the challenges in diagnosing acute HIV infection without the use of viral load testing in this rare elite controller phenotype.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs) increase the level of viral transcription from ...cells producing HIV RNA or harboring transcriptionally-inactive (latent) infection. We therefore developed a microfluidic single-cell-in-droplet (scd)PCR assay to directly measure the number of CD4+ T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin) or T cell receptor (TCR) stimulation. Detection of HIV-1 transcriptional activity can also be performed on hundreds of thousands of CD4+ T-cells in a single experiment. The scdPCR method was then applied to CD4+ T cells obtained from HIV-1-infected individuals on antiretroviral therapy. Overall, our results suggest that effects of LRAs on HIV-1 reactivation may be heterogeneous—increasing transcription from active cells in some cases and increasing the number of transcriptionally active cells in others. Genomic DNA and human mRNA isolated from HIV-1 reactivated cells could also be detected and quantified from individual cells. As a result, our assay has the potential to provide needed insight into various reservoir eradication strategies.
•A common approach to HIV cure involves reactivating HIV-infected cells.•Developed single cell assay to directly quantify HIV transcriptionally reactivated cells.•Single cell effects of latency reversing agents on HIV reactivation are heterogeneous.•Bulk cell-associated HIV RNA levels are divergent from the number of RNA-producing cells.•Assay allows for single-cell quantification of genomic DNA and mRNA following target cell identification.
We designed and implemented a microfluidic, single-cell assay to directly measure the number of individual cells from individuals on antiretroviral therapy that produce HIV-1 RNA. The assay also allows for single-cell quantification of human genomic DNA and messenger RNA following identification and isolation of actively HIV-1-infected cells. The ability to directly measure transcriptional activity of HIV-1 in individual cells followed by downstream characterization of human and viral genetic information within these cells has the potential to provide a greater mechanistic understanding of experimental strategies to purge residual HIV-1 reservoirs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The aim of this study was to assess soluble CD30 (sCD30), a protein that colocalises with HIV-1 RNA and DNA in lymphoid cells and tissues, in cerebrospinal fluid (CSF) as a marker of HIV-1 infection ...in the central nervous system (CNS).
This was a cross-sectional study using archived samples from two clinical cohorts. Soluble CD30 concentrations were measured in paired CSF and plasma from untreated viraemic individuals (
=52), individuals on suppressive antiretroviral therapy (ART) (
=33), HIV-1 controllers (
=10), participants with CSF HIV-1 'escape' (
=11) and controls without HIV-1 infection (
=16). Nonparametric tests were used to compare levels across groups and evaluate correlations with HIV-1 RNA, CSF neurofilament light chain protein (NFL) and neopterin.
Compared with controls (median 30 ng/mL, interquartile range IRQ 23-50), plasma sCD30 levels were elevated in viraemic participants (75 ng/mL, 52-116;
<0.001), but not in those on suppressive ART (38 ng/mL, 32-62). In contrast, CSF sCD30 levels were elevated in ART-suppressed individuals (34 ng/mL, 19-46;
=0.001) and in those with CSF 'escape' (33 ng/mL, 27-40;
=0.004) compared with controls (18 ng/mL, 11-23), but not in untreated viraemic individuals. No association was observed between CSF sCD30 and plasma HIV-1 RNA, concurrent or nadir CD4+ T cell count, duration of infection or plasma sCD30. CSF sCD30 correlated with CSF NFL (
=0.34,
=0.001).
In contrast to plasma, sCD30 levels are elevated in the CSF of individuals with HIV-1 infection who are on suppressive ART. Elevated levels of sCD30 in the CSF may be an indicator of persistent CNS HIV-1 infection, although the mechanism underlying this elevation warrants further investigation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Pharmacologic inhibition of the mammalian target of rapamycin (mTOR) in the setting of renal transplantation has previously been associated with lower human immunodeficiency virus 1 (HIV‐1) DNA ...burden, and in vitro studies suggest that mTOR inhibition may lead to HIV transcriptional silencing. Because prospective clinical trials are lacking, we conducted an open‐label, single‐arm study to determine the impact of the broad mTOR inhibitor, everolimus, on residual HIV burden, transcriptional gene expression profiles, and immune responses in HIV‐infected adult solid organ transplant (SOT) recipients on antiretroviral therapy. Whereas everolimus therapy did not have an overall effect on cell‐associated HIV‐1 DNA and RNA levels in the entire cohort, participants who maintained everolimus time‐averaged trough levels >5 ng/mL during the first 2 months of therapy had significantly lower RNA levels up to 6 months after the cessation of study drug. Time‐averaged everolimus trough levels significantly correlated with greater inhibition of mTOR gene pathway transcriptional activity. Everolimus treatment also led to decreased PD‐1 expression on certain T cell subsets. These data support the rationale for further study of the effects of mTOR inhibition on HIV transcriptional silencing in non‐SOT populations, either alone or in combination with other strategies.
Trial Registration: ClinicalTrials.gov NCT02429869.
Six months of everolimus therapy in solid organ transplant recipients with HIV controlled by antiretroviral therapy did not have an overall effect on HIV reservoir size, but participants who maintained higher everolimus trough levels during the first 2 months had significantly lower cell‐associated HIV‐1 RNA levels that persisted for 6 months after everolimus cessation.
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BFBNIB, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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