Immune cells promote paralytic disease in mice infected with enterovirus D68

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Abstract

Enterovirus D68 (EV-D68) is associated with acute flaccid myelitis (AFM), a poliomyelitis-like illness causing paralysis in young children. However, mechanisms of paralysis are unclear, and antiviral therapies are lacking. To better understand EV-D68 disease, we inoculated newborn mice intracranially to assess viral tropism, virulence, and immune responses. Wild-type (WT) mice inoculated intracranially with a neurovirulent strain of EV-D68 showed infection of spinal cord neurons and developed paralysis. Spinal tissue from infected mice revealed increased chemokines, inflammatory monocytes, macrophages, and T cells relative to controls, suggesting that immune cell infiltration influences pathogenesis. To define the contribution of cytokine-mediated immune cell recruitment to disease, we inoculated mice lacking CCR2, a receptor for several EV-D68-upregulated cytokines, or RAG1, which is required for lymphocyte maturation. WT, Ccr2-/-, and Rag1-/- mice had comparable viral titers in spinal tissue. However, Ccr2-/- and Rag1-/- mice were significantly less likely to be paralyzed relative to WT mice. Consistent with impaired T cell recruitment to sites of infection in Ccr2-/- and Rag1 -/- mice, antibody-mediated depletion of CD4+ or CD8+ T cells from WT mice diminished paralysis. These results indicate that immune cell recruitment to the spinal cord promotes EV-D68-associated paralysis and illuminate new targets for therapeutic intervention.

Authors

Mikal A. Woods Acevedo, Jie Lan, Sarah Maya, Jennifer E. Jones, Isabella E. Bosco, John V. Williams, Megan C. Freeman, Terence S. Dermody

Widespread distribution of transcriptionally active, clonally expanded, HIV-1 proviruses despite suppressive antiretroviral therapy

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Abstract

The rapid viral rebound observed following treatment interruption, despite prolonged time on antiretroviral therapy with plasma HIV-RNA levels <40 copies/mL, suggests persistent HIV-1 reservoir(s) outside of the blood. Studies of HIV-1 proviruses in autopsy tissue samples have hinted at their persistence. However, their distribution across different anatomical compartments and their transcriptional activity within tissues remains unclear. The present study has examined molecular DNA and RNA reservoirs of HIV-1 in autopsy samples from 13 individuals with HIV-1 infection. Of the 13, 5 had detectable levels of HIV-1 RNA in plasma while 8 did not. Cell associated HIV-RNA was detected in 12 out of 13 donors and in 27 of the 30 different tissues examined. HIV-specific DNA and RNA were widely distributed and predominantly associated with clonal expansions. No significant differences were noted between the groups and no tissues were preferentially affected. These data imply that a substantial seeding of tissues with cells harboring transcriptionally active proviral DNA can be seen in the setting of HIV-1 infection despite ART and highlight one of the challenges in achieving an HIV-1 cure.

Authors

Hiromi Imamichi, Ven Natarajan, Francesca Scrimieri, Mindy Smith, Yunden Badralmaa, Marjorie Bosche, Jack M. Hensien, Thomas Buerkert, Weizhong Chang, Brad T. Sherman, Kanal Singh, H. Clifford Lane

Activating antiviral immune responses potentiates immune checkpoint inhibition in glioblastoma models

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Abstract

Viral mimicry refers to the activation of innate antiviral immune responses due to the induction of endogenous retroelements (REs). Viral mimicry augments antitumor immune responses and sensitizes solid tumors to immunotherapy. Here, we found that targeting what we believe to be a novel, master epigenetic regulator, Zinc Finger Protein 638 (ZNF638), induces viral mimicry in glioblastoma (GBM) preclinical models and potentiates immune checkpoint inhibition (ICI). ZNF638 recruits the HUSH complex, which precipitates repressive H3K9me3 marks on endogenous REs. In GBM, ZNF638 is associated with marked locoregional immunosuppressive transcriptional signatures, reduced endogenous RE expression, and poor immune cell infiltration. Targeting ZNF638 decreased H3K9 trimethylation, increased REs, and activated intracellular dsRNA signaling cascades. Furthermore, ZNF638 knockdown upregulated antiviral immune programs and significantly increased PD-L1 immune checkpoint expression in diverse GBM models. Importantly, targeting ZNF638 sensitized mice to ICI in syngeneic murine orthotopic models through innate IFN signaling. This response was recapitulated in recurrent GBM (rGBM) samples with radiographic responses to checkpoint inhibition with widely increased expression of dsRNA, PD-L1, and perivascular CD8 cell infiltration, suggesting that dsRNA signaling may mediate response to immunotherapy. Finally, low ZNF638 expression was a biomarker of clinical response to ICI and improved survival in patients with rGBM and patients with melanoma. Our findings suggest that ZNF638 could serve as a target to potentiate immunotherapy in gliomas.

Authors

Deepa Seetharam, Jay Chandar, Christian K. Ramsoomair, Jelisah F. Desgraves, Alexandra Alvarado Medina, Anna Jane Hudson, Ava Amidei, Jesus R. Castro, Vaidya Govindarajan, Sarah Wang, Yong Zhang, Adam M. Sonabend, Mynor J. Mendez Valdez, Dragan Maric, Vasundara Govindarajan, Sarah R. Rivas, Victor M. Lu, Ritika Tiwari, Nima Sharifi, Emmanuel Thomas, Marcus Alexander, Catherine DeMarino, Kory Johnson, Macarena I. De La Fuente, Ruham Alshiekh Nasany, Teresa Maria Rosaria Noviello, Michael E. Ivan, Ricardo J. Komotar, Antonio Iavarone, Avindra Nath, John Heiss, Michele Ceccarelli, Katherine B. Chiappinelli, Maria E. Figueroa, Defne Bayik, Ashish H. Shah

Divergent populations of HIV-infected naïve and memory CD4+ T-cell clones in children on antiretroviral therapy

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Abstract

BACKGROUND. Naïve cells comprise 90% of the CD4+ T-cell population in neonates and exhibit distinct age-specific capacities for proliferation and activation. We hypothesized that HIV-infected naïve CD4+ T-cell populations in children on long-term antiretroviral therapy (ART) would thus be distinct from infected memory cells. METHODS. Peripheral blood naïve and memory CD4+ T cells from 8 children with perinatal HIV on ART initiated at age 1.7-17 months were isolated by FACS. DNA was extracted from sorted cells and HIV proviruses counted, evaluated for intactness, and subjected to integration site analysis. RESULTS. Naïve CD4+ T cells containing HIV proviruses were detected in children with 95% statistical confidence. A median of 4.7% of LTR-containing naïve CD4+ T cells also contained HIV genetic elements consistent with intactness. Full-length proviral sequencing confirmed intactness of one provirus. In the participant with the greatest level of naïve cell infection, ISA revealed infected expanded cell clones in both naïve and memory T cells with no common HIV integration sites detected between subsets. Divergent integration site profiles reflected differential gene expression patterns of naïve and memory T cells. CONCLUSIONS. These results demonstrate that HIV persists in both naïve and memory CD4+ T cells that undergo clonal expansion and harbor intact proviruses, suggesting that infected memory T-cell clones do not frequently arise from naïve cell differentiation in children with perinatal HIV on long-term ART. FUNDING. Center for Cancer Research, NCI and Office of AIDS Research funding to MFK, NCI FLEX funding to JWR. Children’s and Emory JFF pilot to MM.

Authors

Mary Grace Katusiime, Victoria Neer, Shuang Guo, Sean C. Patro, Wenjie Wang, Brian Luke, Adam A. Capoferri, Xiaolin Wu, Anna M. Horner, Jason W. Rausch, Ann Chahroudi, Maud Mavigner, Mary F. Kearney

PGLYRP2 drives hepatocyte-intrinsic innate immunity by trapping and clearing hepatitis B virus

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Abstract

Spontaneous clearance of hepatitis B virus (HBV) is frequent in adults (95%) but rare in infants (5%), emphasizing the critical role of age-related hepatic immunocompetence. However, the underlying mechanisms of hepatocyte-specific immunosurveillance and age-dependent HBV clearance remain unclear. Here, we identified PGLYRP2 as a hepatocyte-specific pattern recognition receptor with age-dependent expression, and demonstrated that phase separation of PGLYRP2 was a critical driver of spontaneous HBV clearance in hepatocytes. Mechanistically, PGLYRP2 recognized and potentially eliminated covalently closed circular DNA (cccDNA) via phase separation, coordinated by its intrinsically disordered region and HBV DNA-binding domain (PGLYRP2IDR/209-377) in the nucleus. Additionally, PGLYRP2 suppressed HBV capsid assembly by directly interacting with the viral capsid, mediated by its PGRP domain. This interaction promoted the nucleocytoplasmic translocation of PGLYRP2 and subsequent secretion of the PGLYRP2-HBV capsid complex, thereby bolstering the hepatic antiviral response. Pathogenic variants or deletions in PGLYRP2 impaired its ability to inhibit HBV replication, highlighting its essential role in hepatocyte-intrinsic immunity. These findings suggest that targeting the PGLYRP2-mediated host-virus interaction may offer a potential therapeutic strategy for the development of anti-HBV treatments, representing a promising avenue for achieving a functional cure for HBV infection.

Authors

Ying Li, Huihui Ma, Yongjian Zhang, Tinghui He, Binyang Li, Haoran Ren, Jia Feng, Jie Sheng, Kai Li, Yu Qian, Yunfeng Wang, Haoran Zhao, Jie He, Huicheng Li, Hongjin Wu, Yuanfei Yao, Ming Shi

Integrative analysis reveals therapeutic potential of pyrvinium pamoate in Merkel cell carcinoma

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Abstract

Merkel Cell Carcinoma (MCC) is an aggressive neuroendocrine cutaneous malignancy arising from either ultraviolet-induced mutagenesis or Merkel cell polyomavirus (MCPyV) integration. Despite extensive research, our understanding of the molecular mechanisms driving the transition from normal cells to MCC remains limited. To address this knowledge gap, we assessed the impact of inducible MCPyV T antigens on normal human fibroblasts by performing RNA sequencing. Our data uncovered changes in expression and regulation of Wnt signaling pathway members. Building on this observation, we bioinformatically evaluated various Wnt pathway perturbagens for their ability to reverse the MCC gene expression signature and identified pyrvinium pamoate, an FDA-approved anthelminthic drug known for its anti-tumor activity in other cancers. Leveraging transcriptomic, network, and molecular analyses, we found that pyrvinium targets multiple MCC vulnerabilities. Pyrvinium not only reverses the neuroendocrine features of MCC by modulating canonical and non-canonical Wnt signaling but also inhibits cancer cell growth by activating p53-mediated apoptosis, disrupting mitochondrial function, and inducing endoplasmic reticulum stress. Finally, we demonstrated that pyrvinium reduces tumor growth in an MCC mouse xenograft model. These findings offer a new understanding of the role of Wnt signaling in MCC and highlight the utility of pyrvinium as a potential treatment for MCC.

Authors

Jiawen Yang, James T. Lim, Paul Victor Santiago Raj, Marcelo G. Corona, Chen Chen, Hunain Khawaja, Qiong Pan, Gillian D. Paine-Murrieta, Rick G. Schnellmann, Denise J. Roe, Prafulla C. Gokhale, James A. DeCaprio, Megha Padi

Transcription of hepatitis B surface antigen shifts from cccDNA to integrated HBV DNA during treatment

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Abstract

The cornerstone of functional cure for chronic hepatitis B (CHB) is hepatitis B surface antigen (HBsAg) loss from blood. HBsAg is encoded by covalently closed circular DNA (cccDNA) and HBV DNA integrated into the host genome (iDNA). Nucleos(t)ide analogues (NUCs), the mainstay of CHB treatment, rarely lead to HBsAg loss, which we hypothesized was due to continued iDNA transcription despite decreased cccDNA transcription. To test this, we applied a novel multiplex droplet digital PCR that identifies the dominant source of HBsAg mRNAs to 3436 single cells from paired liver biopsies from ten people with CHB and HIV receiving NUCs. With increased NUC duration, cells producing HBsAg mRNAs shifted from chiefly cccDNA to chiefly iDNA. This shift was due to both a reduction in the number of cccDNA-containing cells and diminished cccDNA-derived transcription per cell; furthermore, it correlated with reduced detection of proteins deriving from cccDNA but not iDNA. Despite this shift in the primary source of HBsAg, rare cells remained with detectable cccDNA-derived transcription, suggesting a source for maintaining the replication cycle. Functional cure must address both iDNA and residual cccDNA transcription. Further research is required to understand the significance of HBsAg when chiefly derived from iDNA.

Authors

Maraake Taddese, Tanner Grudda, Giulia Belluccini, Mark Anderson, Gavin Cloherty, Hyon S. Hwang, Monika Mani, Che-Min Lo, Naomi Esrig, Mark S. Sulkowski, Richard K. Sterling, Yang Zhang, Ruy M. Ribeiro, David L. Thomas, Chloe L. Thio, Ashwin Balagopal

Senescence of endothelial cells increases susceptibility to Kaposi's sarcoma-associated herpesvirus infection via CD109-mediated viral entry

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Abstract

The aging process is characterized by cellular functional decline and increased susceptibility to infections. Understanding the association between virus infection and aging is crucial for developing effective strategies against viral infections in older individuals. However, the relationship between Kaposi's sarcoma-associated herpesvirus (KSHV) infection, a cause of Kaposi's sarcoma prevalent among the elderly without HIV infection, and cellular senescence remains enigmatic. This study uncovers a fascinating link between cellular senescence and enhanced KSHV infectivity in human endothelial cells. Through a comprehensive proteomic analysis, we identified caveolin-1 and CD109 as novel host factors significantly upregulated in senescent cells that promote KSHV infection. Remarkably, CRISPR-Cas9-mediated knockout of these factors reduced KSHV binding and entry, leading to decreased viral infectivity. Furthermore, surface plasmon resonance analysis and confocal microscopy revealed a direct interaction between KSHV virions and CD109 on the cell surface during entry, with recombinant CD109 protein exhibiting an intriguing ability to inhibit infection by blocking virion binding. These findings uncover a previously unrecognized role of cellular senescence in enhancing KSHV infection through upregulation of specific host factors and provide novel insights into the complex interplay between aging and viral pathogenesis.

Authors

Myung-Ju Lee, Jun-Hee Yeon, Jisu Lee, Yun Hee Kang, Beom Seok Park, Joo Hee Park, Sung-Ho Yun, Dagmar Wirth, Seung-Min Yoo, Changhoon Park, Shou-Jiang Gao, Myung-Shin Lee

Assessing advances in three decades of clinical antiretroviral therapy on the HIV-1 reservoir

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Abstract

BACKGROUND. Antiretroviral therapy (ART) has improved the clinical management of HIV-1 infection. However, little is known about how the latest ART recommendations affect the heterogeneity of HIV-1 reservoir size. METHODS. We used a complete statistical approach to outline parameters underlying diversity in HIV-1 reservoir size in a cohort of 892 people with HIV-1 (PWH) on suppressive ART for >3 years. Total HIV-1-DNA levels were measured in PBMCs using digital droplet PCR (ddPCR). RESULTS. We classified 179 (20%) participants as Low Viral Reservoir Treated (LoViReT, <50 HIV-1-DNA copies/106 PBMCs). Twenty variables were collected to explore their association with the LoViReT phenotype using machine learning approaches. Nadir CD4 and zenith pre-ART viral load were closely associated with LoViReT status, with lower CD4 recovery, shorter time from diagnosis to undetectable viral load, and initiation of treatment with an integrase inhibitor (InSTI)–containing regimen. Initiating ART with any InSTI was also associated with shorter time to undetectable viremia. Locally estimated scatterplot smoothing (LOESS) regression revealed a progressive reduction in the size of the HIV-1 reservoir in individuals who started ART after 2007. Similarly, higher nadir CD4 and shorter time to undetectable viremia were observed when treatment was initiated after that year. CONCLUSION. Our findings demonstrate that the progressive implementation of earlier, universal treatment at diagnosis and the use of InSTIs affect the size of the HIV-1 reservoir. Our work shows that effective management of infection is the first step toward reducing the reservoir and brings us closer to achieving a cure. FUNDING. U.S. National Institutes of Health, Division of AIDS at the National Institute of Allergy and Infectious Diseases, Merck Sharp & Dohme.

Authors

Irene González-Navarro, Víctor Urrea, Cristina Gálvez, Maria del Carmen Garcia-Guerrero, Sara Morón-López, Maria C. Puertas, Eulàlia Grau, Beatriz Mothe, Lucía Bailón, Cristina Miranda, Felipe García, Lorna Leal, Linos Vandekerckhove, Vincent C. Marconi, Rafick P. Sekaly, Bonaventura Clotet, Javier Martinez-Picado, Maria Salgado

A large-scale population-based study reveals that gp42-IgG antibody is protective against Epstein-Barr virus-associated nasopharyngeal carcinoma

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Abstract

BACKGROUND. Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC), but the existence of NPC protective antibody against EBV-associated antigens remains inconclusive. METHODS. NPC cases and matched controls were identified from prospective cohorts comprising 75,481 participants in southern China. ELISA and conditional logistic regression were applied to assess effects of gp42-IgG on NPC. The expression of HLA-II, the gp42 receptor, in nasopharyngeal atypical dysplasia and its impact on EBV infecting epithelial cells were evaluated. FINDINGS. gp42-IgG titers were significantly lower in NPC cases compared to controls across various follow-up years before NPC diagnosis (P<0.05). Individuals in the highest quartile of gp42-IgG titers had a 71% NPC risk reduction comparing to those in the lowest quartile (odds ratios [OR]Q4vsQ1=0.29, 95% confidence intervals [CIs]=0·15 to 0·55, P<0.001). Each unit antibody titer increase was associated with 34% lower risk of NPC (OR=0.66, 95% CI=0.54 to 0.81, Ptrend <0.001). Their protective effect was observed in cases diagnosed ≥5 years, 1-5 years and <1 year after blood collection (P<0.05). HLA-II expression was detected in 13 of 27 nasopharyngeal atypical dysplasia and its overexpression substantially promoted epithelial-cell-origin EBV infection. CONCLUSION. Elevated EBV gp42-IgG titers can reduce NPC risk, indicating gp42 as a potential EBV prophylactic vaccine design target. TRIAL REGISTRATION. NCT00941538, NCT02501980, ChiCTR2000028776, ChiCTR2100041628. FUNDING. Noncommunicable Chronic Diseases-National Science and Technology Major Project, National Natural Science Foundation of China, Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program, Central Financial Transfer Payment Projects of the Chinese Government, Cancer Research Grant of Zhongshan City.

Authors

Xiang-Wei Kong, Guo-Long Bu, Hua Chen, Yu-Hua Huang, Zhiwei Liu, Yin-Feng Kang, Yan-Cheng Li, Xia Yu, Biao-Hua Wu, Zi-Qian Li, Xin-Chun Chen, Shang-Hang Xie, Dong-Feng Lin, Tong Li, Shu-Mei Yan, Run-Kun Han, Nan Huang, Qian-Yu Wang, Yan Li, Ao Zhang, Qian Zhong, Xiao-Ming Huang, Weimin Ye, Ming-Fang Ji, Yong-Lin Cai, Su-Mei Cao, Mu-Sheng Zeng