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
It is unknown if extremely early initiation of antiretroviral therapy (ART) may lead to long-term ART-free HIV remission or cure. As a result, we studied 2 individuals recruited from a pre-exposure ...prophylaxis (PrEP) program who started prophylactic ART an estimated 10 days (Participant A; 54-year-old male) and 12 days (Participant B; 31-year-old male) after infection with peak plasma HIV RNA of 220 copies/mL and 3,343 copies/mL, respectively. Extensive testing of blood and tissue for HIV persistence was performed, and PrEP Participant A underwent analytical treatment interruption (ATI) following 32 weeks of continuous ART.
Colorectal and lymph node tissues, bone marrow, cerebral spinal fluid (CSF), plasma, and very large numbers of peripheral blood mononuclear cells (PBMCs) were obtained longitudinally from both participants and were studied for HIV persistence in several laboratories using molecular and culture-based detection methods, including a murine viral outgrowth assay (mVOA). Both participants initiated PrEP with tenofovir/emtricitabine during very early Fiebig stage I (detectable plasma HIV-1 RNA, antibody negative) followed by 4-drug ART intensification. Following peak viral loads, both participants experienced full suppression of HIV-1 plasma viremia. Over the following 2 years, no further HIV could be detected in blood or tissue from PrEP Participant A despite extensive sampling from ileum, rectum, lymph nodes, bone marrow, CSF, circulating CD4+ T cell subsets, and plasma. No HIV was detected from tissues obtained from PrEP Participant B, but low-level HIV RNA or DNA was intermittently detected from various CD4+ T cell subsets. Over 500 million CD4+ T cells were assayed from both participants in a humanized mouse outgrowth assay. Three of 8 mice infused with CD4+ T cells from PrEP Participant B developed viremia (50 million input cells/surviving mouse), but only 1 of 10 mice infused with CD4+ T cells from PrEP Participant A (53 million input cells/mouse) experienced very low level viremia (201 copies/mL); sequence confirmation was unsuccessful. PrEP Participant A stopped ART and remained aviremic for 7.4 months, rebounding with HIV RNA of 36 copies/mL that rose to 59,805 copies/mL 6 days later. ART was restarted promptly. Rebound plasma HIV sequences were identical to those obtained during acute infection by single-genome sequencing. Mathematical modeling predicted that the latent reservoir size was approximately 200 cells prior to ATI and that only around 1% of individuals with a similar HIV burden may achieve lifelong ART-free remission. Furthermore, we observed that lymphocytes expressing the tumor marker CD30 increased in frequency weeks to months prior to detectable HIV-1 RNA in plasma. This study was limited by the small sample size, which was a result of the rarity of individuals presenting during hyperacute infection.
We report HIV relapse despite initiation of ART at one of the earliest stages of acute HIV infection possible. Near complete or complete loss of detectable HIV in blood and tissues did not lead to indefinite ART-free HIV remission. However, the small numbers of latently infected cells in individuals treated during hyperacute infection may be associated with prolonged ART-free remission.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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
Abstract Assays that can verify full viral eradication are essential in the context of achieving a cure for HIV/AIDS. In vitro quantitative viral out growth assays (qVOA) are currently the gold ...standard for measuring latent HIV-1 but these assays often fail to detect very low levels of replication-competent virus. Here we investigated an alternative in vivo approach for sensitive viral detection using humanized mice (hmVOA). Peripheral blood CD4+ T cell samples from HIV subjects on stable ART with undetectable viral loads by RT-PCR were first assayed by in vitro qVOA. Corresponding patient samples in which no virus was detected by qVOA were injected into humanized mice to allow viral outgrowth. Of the five qVOA virus negative samples, four gave positive viral outgrowth in the hmVOA assay suggesting that it is more sensitive in detecting latent HIV-1.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Human Herpes Virus 8 (HHV8) can cause Kaposi's Sarcoma (KS) in immunosuppressed individuals. However, little is known about the association between chemotherapy or hematopoietic stem cell ...transplantation (HSCT), circulating HHV8 DNA levels, and clinical KS in HIV-1-infected individuals with various malignancies. Therefore, we examined the associations between various malignancies, systemic cancer chemotherapy, T cell phenotypes, and circulating HHV8 DNA in 29 HIV-1-infected participants with concomitant KS or other cancer diagnoses.
We quantified HHV8 plasma viral loads and cell-associated HHV8 DNA and determined the relationship between circulating HHV8 DNA and lymphocyte counts, and markers of early and late lymphocyte activation, proliferation and exhaustion.
There were no significant differences in plasma HHV8 DNA levels between baseline and post-chemotherapy time points or with the presence or absence of clinical KS. However, in two participants circulating HHV8 DNA increased following treatment for KS or HSCT for lymphoma,. We observed an approximately 2-log10 reduction in plasma HHV8 DNA in an individual with KS and multicentric Castleman disease following rituximab monotherapy. Although individuals with clinical KS had lower mean CD4+ T cell counts and percentages as expected, there were no significant associations with these factors and plasma HHV8 levels. We identified increased proportions of CD8+ and CD4+ T cells expressing CD69 (P = 0.01 & P = 0.04 respectively), and increased CD57 expression on CD4+ T cells (P = 0.003) in participants with detectable HHV8.
These results suggest there is a complex relationship between circulating HHV8 DNA and tissue-based disease in HIV-1 and HHV8 co-infected individuals with various malignancies.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of ...detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients’ homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE²RD), which addresses all these impediments on a single platform. The NE²RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE²RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE²RD’s broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the pointof-care or primary care settings and at patients’ homes.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Background. Systemic chemotherapies for various malignancies have been shown to significantly, yet transiently, decrease numbers of CD4+ T lymphocytes, a major reservoir for human immunodeficiency ...virus type 1 (HIV-1) infection. However, little is known about the impact of cytoreductive chemotherapy on HIV-1 reservoir dynamics, persistence, and immune responses. Methods. We investigated the changes in peripheral CD4+ T-cell–associated HIV-1 DNA and RNA levels, lymphocyte activation, viral population structure, and virus-specific immune responses in a longitudinal cohort of 15 HIV-1–infected individuals receiving systemic chemotherapy or subsequent autologous stem cell transplantation for treatment of hematological malignancies and solid tumors. Results. Despite a transient reduction in CD4+ T cells capable of harboring HIV-1, a 1.7- and 3.3-fold increase in mean CD4+ T-cell–associated HIV-1 RNA and DNA, respectively, were observed months following completion of chemotherapy in individuals on antiretroviral therapy. We also observed changes in CD4+ T-cell population diversity and clonal viral sequence expansion during CD4+ T-cell reconstitution following chemotherapy cessation. Finally, HIV-1 DNA was preferentially, and in some cases exclusively, detected in cytomegalovirus (CMV)– and Epstein-Barr virus (EBV)–responsive CD4+ T cells following chemotherapy. Conclusions. Expansion of HIV-infected CMV/EBV-specific CD4 + T cells may contribute to maintenance of the HIV DNA reservoir following chemotherapy.
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BFBNIB, NMLJ, NUK, PNG, SAZU, 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
Data are limited regarding adverse reactions after COVID-19 vaccination in patients with a history of multisystem inflammatory syndrome in children (MIS-C). The lack of vaccine safety data in this ...unique population may cause hesitancy and concern for many families and health care professionals.
To describe adverse reactions following COVID-19 vaccination in patients with a history of MIS-C.
In this multicenter cross-sectional study including 22 North American centers participating in a National Heart, Lung, and Blood Institute, National Institutes of Health-sponsored study, Long-Term Outcomes After the Multisystem Inflammatory Syndrome in Children (MUSIC), patients with a prior diagnosis of MIS-C who were eligible for COVID-19 vaccination (age ≥5 years; ≥90 days after MIS-C diagnosis) were surveyed between December 13, 2021, and February 18, 2022, regarding COVID-19 vaccination status and adverse reactions.
COVID-19 vaccination after MIS-C diagnosis.
The main outcome was adverse reactions following COVID-19 vaccination. Comparisons were made using the Wilcoxon rank sum test for continuous variables and the χ2 or Fisher exact test for categorical variables.
Of 385 vaccine-eligible patients who were surveyed, 185 (48.1%) received at least 1 vaccine dose; 136 of the vaccinated patients (73.5%) were male, and the median age was 12.2 years (IQR, 9.5-14.7 years). Among vaccinated patients, 1 (0.5%) identified as American Indian/Alaska Native, non-Hispanic; 9 (4.9%) as Asian, non-Hispanic; 45 (24.3%) as Black, non-Hispanic; 59 (31.9%) as Hispanic or Latino; 53 (28.6%) as White, non-Hispanic; 2 (1.1%) as multiracial, non-Hispanic; and 2 (1.1%) as other, non-Hispanic; 14 (7.6%) had unknown or undeclared race and ethnicity. The median time from MIS-C diagnosis to first vaccine dose was 9.0 months (IQR, 5.1-11.9 months); 31 patients (16.8%) received 1 dose, 142 (76.8%) received 2 doses, and 12 (6.5%) received 3 doses. Almost all patients received the BNT162b2 vaccine (347 of 351 vaccine doses 98.9%). Minor adverse reactions were observed in 90 patients (48.6%) and were most often arm soreness (62 patients 33.5%) and/or fatigue (32 17.3%). In 32 patients (17.3%), adverse reactions were treated with medications, most commonly acetaminophen (21 patients 11.4%) or ibuprofen (11 5.9%). Four patients (2.2%) sought medical evaluation, but none required testing or hospitalization. There were no patients with any serious adverse events, including myocarditis or recurrence of MIS-C.
In this cross-sectional study of patients with a history of MIS-C, no serious adverse events were reported after COVID-19 vaccination. These findings suggest that the safety profile of COVID-19 vaccination administered at least 90 days following MIS-C diagnosis appears to be similar to that in the general population.
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