Viral transmission and pathogenesis in human tissues

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications, trials & patents

Abstract

1. Cytokines in HIV disease We followed longitudinally 90 people living with HIV (PWH) (59 women and 31 men) already virally suppressed and on antiretroviral therapy (ART) on average for 6 years. We measured the concentrations of 41 cytokines in their blood plasma and followed their trajectory overtime. We found that long ART had a significant effect on the trajectory of 9 cytokines. In particular, the concentration of 8 cytokines continued to decrease over time (CD163; IP-10; CXCL13; I-TAC; MCP-1; MIG; MIP-3 and TNF-), while LBP concentration slightly increased over time. Our results suggest that long ART continues to modulate pro-inflammatory cytokines past the initial decrease in concentrations observed during ART suppressive therapy when viral load becomes undetectable. 2. Extracellular vesicle-associated cytokines in non-progressors among PWH We evaluated the cytokine profile associated with extracellular vesicles (EVs) in plasma samples from well-characterized cohorts of PWH with different virological control status. PWH were divided into 5 groups: ART-nave; ART-treated with undetectable viremia; elite controllers (EC) who controlled viremia (both transient controllers (TC) and persistent controllers (PC)); and uninfected individuals. Levels of 39 cytokines within and on the surface of EVs isolated from their stored plasma were quantified. Random forest, principal component analysis, and decision tree analyses were performed to identify specific cytokines as potential signatures of each study group. EC showed the highest levels of EV-associated cytokines, especially when PC were compared to TC. Higher levels of EV IL-18 were identified as a biomarker for EC population in the context of suppressed viremia, and higher levels of EV IL-3 and TRAIL were best at discriminating the PC phenotype from TC. In conclusion, higher levels of EV-associated cytokines in plasma may represent a new signature for the EC population. 3. EVs in immune activation of HIV-infected tissues under ART We investigated the mechanisms by which EVs from HIV-1-infected/ART-suppressed lymphoid tissues lead to immune activation. Using fluorescently labeled EVs and flow cytometry, we identified B cells as a prime target cell population of EVs from these tissues. Over time, the amount of pre-germinal center B cells increased followed by increased germinal center B cells, and B cell activation markers (CD40, CD80, CD86). By day 9, effector memory T cells increased, and natural killer cells increased activation markers (CD38, CD69, CD80, CD86, HLA-DR). The increase in cytokines was traced to multiple cell types including CD4 T cells (MIP-1a, MIP-1b, RANTES), CD8 T cells (IFN-g, MIP-1a, MIP-1b, RANTES, TNFa), B cells (MIP-1a, MIP-1b, RANTES), NK cells (RANTES) and monocytes (RANTES). Understanding the cascade of events triggered by EVs from may lead to potential therapeutic targets for suppression of improper immune activation in HIV-infected individuals under ART. 4. Localization of an HIV-1 protein on EVs We used several techniques to determine the localization of Nef in EVs. All approaches, including vesicle permeabilization, stripping of surface proteins, and remixing of EVs with Nef, led to the same conclusion: over 90% of the Nef molecules in the EV particles were located at the surface of the EVs. This result is consistent with Nef incorporation in the EVs via accession of the maturing late endosomes and/or the multivesicular bodies, and represent molecules associated with EVs during MVB fusion with the plasma membrane. Although only about 0.5% of total secreted Nef was associated with EVs, the Nef EVs were more biologically potent than soluble Nef. The distribution of Nef in EVs isolated from Nef-transfected cells was confirmed in EVs isolated from lymphocytes from PWH. Our study suggests that Nef can be released from cells in two forms: the dominant form is free protein, and the minor form is Nef associated with EVs with high bioactivity. Our results suggest that Nef EVs can be targeted by immunotherapy to treat HIV-associated co-morbidities. 5. EV-associated cytokines in complicated pregnancies leading to fetal death We found five proteins (CXCL6, endoglin, MIP-3, PlGF, and TFPI) to be different in concentrations in cases of fetal death complicated by preeclampsia compared to those without preeclampsia. Our data suggest that mechanisms of fetal death complicated with preeclampsia include the dysregulation of angiogenic, coagulation and inflammatory pathways. Among cases of fetal death, unsupervised clustering based on the proteins differentially expressed in either the EV or soluble fractions of patients with fetal death relative to controls revealed three major clusters of patients with distinct clinical and placental histopathological characteristics. Cluster 1 included 19 cases of fetal death that had 33 differentially expressed proteins in either the EV or the soluble fraction. Many of these cases of fetal death had two or more features consistent with maternal vascular malperfusion (MVM) and preeclampsia. Cluster 2 contained 12 cases of fetal death, and only PDL1 in the soluble fraction had different concentrations in this subgroup compared to controls. Fewer cases involved MVM lesions or preeclampsia but included the highest percentage of unexplained fetal death cases. Cluster 3 included 16 cases of fetal death with 21 proteins differentially expressed in either the EV or the soluble fraction of patients compared to the controls and fewer features of MVM and preeclampsia than cluster 1. Overall, our results demonstrate the value of including EV-associated biomarkers to improve the prediction of fetal death and understand the pathophysiology of this obstetrical syndrome. 6. Blocking SARS-CoV-2 transmission Nanocellulose particles were obtained from ivory nuts from a palm tree from the Arecaceae family that grows in tropical regions of South America and modified with dense sulfate charges. Due to its derivation from the endosperm of the palms seed, it was designated endospermic nanocellulose (ENC). The unique primary cell walls yielded more homogeneous and thinner cellulose nanoparticles compared to cell walls from other vegetal tissues. We evaluated the feasibility of sulfonated ENC as an inhibitor of viral transmission first by ELISA, which demonstrated effective blocking of SARS-CoV-2 S protein interaction with ACE2 in the presence of ENC, and allowed us to identify optimal working concentrations of ENC. We then used two different methods of blocking transmission of virus to cell cultures using ENC: a non-specific method of ENC encapsulation of SARS-CoV-2 pseudovirus particles or 293T-ACE2 target cells, and a highly specific method immobilizing molecular lures, anti-SARS-CoV-2 spike (S) protein antibodies and soluble ACE2 receptors, in ENC to bind virus. Both methods demonstrated that ENC effectively blocked cell infection by SARS-CoV-2 pseudoviruses, demonstrating the universal binding property of ENC and its usefulness for development of new strategies to prevent viral transmission. This material has potential applications as a film for masks and filters, to bind viral particles and increasing the effectiveness of these filtration devices, or to create adsorbent surfaces to capture or inactivate viruses. The concentrations of ENC used in these experiments will lend themselves easily to aerosolization making them ideal for such purposes. Nanoparticles that are deemed safe for in vivo use also have potential to enhance bioavailability of natural compounds, or for drug delivery.