SARS-CoV-2 ORF8 drives osteoclastogenesis in preexisting immune-mediated inflammatory diseases

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Abstract

Patients with immune-mediated inflammatory diseases (IMIDs) like rheumatoid arthritis (RA) are at higher risk for severe COVID-19 and long-term complications in bone health. Emerging clinical evidence demonstrated that SARS-CoV-2 infection reduces bone turnover and promotes bone loss, but the mechanism underlying worsened bone health remains elusive. This study sought to identify specific immune mediators that exacerbated preexisting IMIDs after SARS-CoV-2 exposure. Plasma samples from 4 groups were analyzed: healthy, IMID only, COVID-19 only, and COVID-19 + IMID. Using high-throughput multiplexed proteomics, we profiled 1,500 protein biomarkers and identified 148 unique biomarkers in COVID-19 patients with IMIDs, including elevated inflammatory cytokines (e.g., IL-17F) and bone resorption markers. Long-term circulating SARS-CoV-2 ORF8, a virulence factor for COVID-19, was detected in the COVID + IMID group. RA was one of the most common IMIDs in our study. ORF8 treatment of RA-derived human osteoblasts (RA-hOBs) increased levels of inflammatory (TNF, IL6, CCL2) and bone resorption (RANKL/osteoprotegerin ratio) markers compared with healthy controls. Supernatants from ORF8-treated RA-hOBs drove the differentiation of macrophages into osteoclast-like cells. These findings suggest that SARS-CoV-2 exposure can exacerbate IMIDs through ORF8-driven inflammation and osteoclastogenesis, highlighting potential therapeutic targets for managing COVID-19–induced bone pathologies.

Authors

Ivonne Melano, Tamiris Azamor, Camila C.S. Caetano, Nikki M. Meyer, Chineme Onwubueke, Anabelle Visperas, Débora Familiar-Macedo, Gielenny M. Salem, Brandy-Lee Soos, Cassandra M. Calabrese, Youn Jung Choi, Shuyang Chen, Younho Choi, Xianfang Wu, Zilton Vasconcelos, Suzy A.A. Comhair, Karin Nielsen-Saines, Leonard H. Calabrese, M. Elaine Husni, Jae U. Jung, Nicolas S. Piuzzi, Suan-Sin Foo, Weiqiang Chen

Rapid systemic spread and minimal immune responses following SIVsab intrarectal transmission in African green monkeys

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Abstract

African green monkeys (AGMs) are natural hosts of SIV whose infection does not progress to AIDS. Since early events of infection may be critical to pathogenesis in nonnatural hosts, we investigated early SIV infection in 29 adult male AGMs intrarectally inoculated with SIVsab92018 (SIVsab) and serially sacrificed throughout acute into early chronic infection to understand patterns of viral establishment, dissemination, and their effect on disease progression. Using this model, we showed that foci of virus replication could be detected at the site of inoculation and in the draining lymphatics as early as 1–3 days postinfection (dpi). Furthermore, testing with ultrasensitive assays showed rapid onset of viremia (2–4 dpi). After systemic spread, virus was detected in all tissues surveyed. Multiple transmitted/founder viruses were identified, confirming an optimal challenge dose, while demonstrating a moderate mucosal genetic bottleneck. Resident CD4+ T cells were the initial target cells; other immune cell populations were not significantly altered at the site of entry. Thus, intrarectal SIVsab infection is characterized by swift dissemination of the virus, a lack of major target cell recruitment, and no window of opportunity for interventions to prevent virus dissemination during the earliest stages of infection, similar to intrarectal transmission but different from vaginal transmission in macaques.

Authors

Kevin D. Raehtz, Cuiling Xu, Claire Deleage, Dongzhu Ma, Benjamin B. Policicchio, Egidio Brocca-Cofano, Daniele Piccolo, Kathryn Weaver, Brandon F. Keele, Jacob D. Estes, Cristian Apetrei, Ivona Pandrea

Variation in HIV-1 Tat activity is a key determinant in the establishment of latent infection

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Abstract

Despite effective treatment, Human immunodeficiency virus (HIV) persists in optimally treated people as a transcriptionally silent provirus. Latently infected cells evade the immune system and the harmful effects of the virus, thereby creating a long-lasting reservoir of HIV. To gain a deeper insight into the molecular mechanisms of HIV latency establishment, we constructed a series of HIV-1 fluorescent reporter viruses that distinguish active versus latent infection. We unexpectedly observed that the proportion of active-to-latent infection depended on a limiting viral factor, which created a bottle neck that could be overcome by superinfection of the cell, T cell activation or overexpression of HIV-1 trans activator of transcription (Tat). In addition, we found that tat and rev expression levels vary amongst HIV molecular clones and that tat levels were an important variable in latency establishment. Lower rev levels limited viral protein expression whereas lower Tat levels or mutation of the Tat binding element promoted latent infection that was resistant to reactivation even in fully activated primary T cells. Nevertheless, we found that combinations of latency reversal agents targeting both cellular activation and histone acetylation pathways overcame deficiencies in the Tat-TAR axis of transcription regulation. These results provide additional insight into the mechanisms of latency establishment and inform Tat-centered approaches to cure HIV.

Authors

Francisco Gomez-Rivera, Valeri H. Terry, Cuie Chen, Mark M. Painter, Maria C. Virgilio, Marianne E. Yaple-Maresh, Kathleen L. Collins

Repurposing of lonafarnib as a treatment for SARS-CoV-2 infection

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Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes Coronavirus disease 2019 (COVID-19), has emerged as a global pandemic pathogen with high mortality. While treatments have been developed to reduce morbidity and mortality of COVID-19, more antivirals with broad-spectrum activities are still needed. Here we identified lonafarnib (LNF), a Food and Drug Administration (FDA)-approved drug inhibitor of cellular farnesyltransferase (FTase), as an effective anti-SARS-CoV-2 agent. LNF inhibited SARS-CoV-2 infection and acted synergistically with known anti-SARS antivirals. LNF was equally active against diverse SARS-CoV-2 variants. Mechanistic studies suggested that LNF targeted multiple steps of viral life cycle. Using other structurally diverse FTase inhibitors and LNF-resistant FTase mutant, we demonstrated a key role of FTase in SARS-CoV-2 life cycle. To demonstrate in vivo efficacy, we infected SARS-CoV-2 susceptible humanized mice expressing human angiotensin-converting enzyme 2 (ACE2) and treated them with LNF. LNF at clinically relevant dose suppressed viral titer in the respiratory tract and improved pulmonary pathology and clinical parameters. Our study demonstrated that LNF, an approved oral drug with excellent human safety data, is a promising antiviral against SARS-CoV-2 that warrants further clinical assessment for treatment of COVID-19 and potentially other viral infections.

Authors

Mohsin Khan, Parker Irvin, Seung Bum Park, Hannah M. Ivester, Inna Ricardo-Lax, Madeleine Leek, Ailis Grieshaber, Eun Sun Jang, Sheryl L. Coutermarsh-Ott, Qi Zhang, Nunziata Maio, Jian-Kang Jiang, Bing Li, Wenwei Huang, Amy Q. Wang, Xin Xu, Zongyi Hu, Wei Zheng, Yihong Ye, Tracey Rouault, Charles M. Rice, Irving C. Allen, T. Jake Liang

Profound reduction of HIV-1 reservoir cells over three decades of antiretroviral therapy started in early infancy

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Abstract

HIV-1 reservoir cells persist indefinitely during suppressive antiretroviral therapy (ART) in individuals who acquire infection in adulthood, but little is known about the longitudinal evolution of viral reservoir cells during long-term ART started during early infancy. We studied two fraternal twins who acquired HIV-1 perinatally, started ART at week 10 after birth and remained on ART for 28 years. We observed that the frequency of genome intact proviruses, determined by single-genome near full-length proviral sequencing, declined by approximately 4,000- to 13,000-fold during this period, indicating enhanced decay rates of intact proviruses even after adjusting for dilution effects from somatic growth. Despite analyzing more than one billion PBMC after 28 years of ART in each participant, no intact proviruses were detected in one participant, and one intact provirus was isolated in the other. The longitudinal decline of defective proviruses in the two participants was more similar to proviral decay kinetics reported in individuals who started ART during adulthood; moreover, clonal sequence clusters were readily detectable for defective proviruses but not for intact proviruses after 28 years of ART in the two twins. Together, these data suggest decreased long-term stability and increased immunological vulnerability of intact proviruses during long-term ART started in early infancy.

Authors

Liliana C. Vela, Leah Carrere, Chloe Naasz, Sruthi Kalavacherla, Toong Seng Tan, Lesley de Armas, Ce Gao, Xu G. Yu, Savita G. Pahwa, Katherine Luzuriaga, Mathias Lichterfeld

Tissue resident memory T cells contribute to protection against heterologous SARS-CoV-2 challenge

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Abstract

Widespread vaccination and natural infection have resulted in greatly decreased rates of severe disease, hospitalization and death after subsequent infection or reinfection with SARS-CoV-2. New vaccine formulations are based on circulating strains of virus, which have tended to evolve to more readily transmit human to human and to evade the neutralizing antibody response. An assumption of this approach is that ancestral strains of virus will not recur. Recurrence of these strains could be a problem for individuals not previously exposed to ancestral spike protein by vaccination or infection. Here, we addressed this question by infecting mice with recent SARS-CoV-2 variants and then challenging them with a highly pathogenic mouse-adapted virus closely related to the ancestral Wuhan-1 strain (SARS2-N501YMA30). We found that challenged mice were protected from death and substantial weight loss, even though they generally had low or no neutralizing antibody response to SARS2-N501YMA30 at the time of reinfection. T cell depletion from the previously infected mice did not diminish infection against clinical disease, although it did result in delayed kinetics of virus clearance in the nasal turbinate and in some cases, in the lungs. Levels of tissue resident memory T cells were significantly elevated in the nasal turbinate of previously infected mice compared to mice that had no previous exposure to SARS-CoV-2. However, this phenotype was not seen in lung tissues. Together, these results indicate that the immune response to newly circulating variants afforded protection against re-infection with the ancestral virus that was at least in part T cell based.

Authors

Abby Odle, Meenakshi Kar, Abhishek K. Verma, Alan Sariol, David K. Meyerholz, Mehul S. Suthar, Lok-Yin Roy Wong, Stanley Perlman

Immunological landscape of human lymphoid explants during measles virus infection

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Abstract

In humans, lymph nodes are the primary site of measles virus (MeV) replication. To understand the immunological events that occur at this site, we infected human lymphoid tissue explants using a pathogenic strain of MeV that expresses GFP. We found that MeV infected 5%–15% of cells across donors. Using single-cell RNA-Seq and flow cytometry, we found that while most of the 29 cell populations identified in the lymphoid culture were susceptible to MeV, there was a broad preferential infection of B cells and reduced infection of T cells. Further subsetting of T cells revealed that this reduction may be driven by the decreased infection of naive T cells. Transcriptional changes in infected B cells were dominated by an interferon-stimulated gene (ISG) signature. To determine which of these ISGs were most substantial, we evaluated the proteome of MeV-infected Raji cells by mass spectrometry. We found that IFIT1, IFIT2, IFIT3, ISG15, CXCL10, MX2, and XAF1 proteins were the most highly induced and positively correlated with their expression in the transcriptome. These data provide insight into the immunological events that occur in lymph nodes during infection and may lead to the development of therapeutic interventions.

Authors

Joshua A. Acklin, Aum R. Patel, Andrew P. Kurland, Shu Horiuchi, Arianna S. Moss, Emma J. DeGrace, Satoshi Ikegame, Jillian Carmichael, Shreyas Kowdle, Patricia A. Thibault, Naoko Imai, Hideki Ueno, Benjamin Tweel, Jeffrey R. Johnson, Brad R. Rosenberg, Benhur Lee, Jean K. Lim

Mechanisms and efficacy of small molecule “latency promoting agents” to inhibit HIV reactivation ex vivo

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Abstract

HIV infection cannot be cured due to the persistence of a reservoir of latently infected cells. Furthermore, virally suppressed individuals experience chronic immune activation from ongoing low-level viral expression. Drugs that inhibit HIV transcription and/or reactivation of latent HIV have been proposed as a strategy to reduce HIV-associated immune activation and/or to achieve a functional cure. We evaluated 26 small molecules, both previously reported drugs and new drug candidates, for their ability to act as “latency promoting/silencing agents (LPAs)” that can reduce or prevent HIV expression after T cell activation. Using a panel of RT-ddPCR assays, we measured the progression through HIV transcription and pinpointed the step at which each of those drugs inhibited HIV transcription, with and without prior activation. While some drugs primarily inhibited one or two steps in HIV reactivation, other drugs (CDK inhibitors, splicing inhibitors, tanespimycin, and triptolide) inhibited multiple stages of HIV transcription and blocked the production of supernatant viral RNA. Dinaciclib, AZD4573, and pladienolide B also appeared to inhibit HIV splicing in unstimulated PBMC. By selecting drugs with known mechanisms of action, we specifically identified cellular factors and pathways that may be involved in regulation of HIV expression. These drugs/targets deserve further study in strategies aimed at reducing HIV-associated immune activation or achieving a functional cure.

Authors

Julie Janssens, Peggy Kim, Sun Jin Kim, Adam Wedrychowski, Gayatri N. Kadiyala, Peter W. Hunt, Steven G. Deeks, Joseph K. Wong, Steven A. Yukl

Expansion of the HSV-2–specific T cell repertoire in skin after immunotherapeutic HSV-2 vaccine

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Abstract

The skin at the site of HSV-2 reactivation is enriched for HSV-2–specific T cells. To evaluate whether an immunotherapeutic vaccine could elicit skin-based memory T cells, we studied skin biopsies and HSV-2–reactive CD4+ T cells from PBMCs by T cell receptor (TCR) β chain (TRB) sequencing before and after vaccination with a replication-incompetent whole-virus HSV-2 vaccine candidate (HSV529). The representation of HSV-2–reactive CD4+ TRB sequences from PBMCs in the skin TRB repertoire increased after the first vaccine dose. We found sustained expansion after vaccination of unique, skin-based T cell clonotypes that were not detected in HSV-2–reactive CD4+ T cells isolated from PBMCs. In one participant, a switch in immunodominance occurred with the emergence of a TCR αβ pair after vaccination that was not detected in blood. This TCRαβ was shown to be HSV-2 reactive by expression of a synthetic TCR in a Jurkat-based NR4A1 reporter system. The skin in areas of HSV-2 reactivation possessed an oligoclonal TRB repertoire that was distinct from the circulation. Defining the influence of therapeutic vaccination on the HSV-2–specific TRB repertoire requires tissue-based evaluation.

Authors

Emily S. Ford, Alvason Z. Li, Kerry J. Laing, Lichun Dong, Kurt Diem, Lichen Jing, Koshlan Mayer-Blackwell, Krithi Basu, Mariliis Ott, Jim Tartaglia, Sanjay Gurunathan, Jack L. Reid, Matyas Ecsedi, Aude G. Chapuis, Meei-Li Huang, Amalia S. Magaret, Christine Johnston, Jia Zhu, David M. Koelle, Lawrence Corey

iGATE analysis improves the interpretability of single-cell immune landscape of influenza infection

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Abstract

Influenza poses a persistent health burden worldwide. To design equitable vaccines effective across all demographics, it is essential to better understand how host factors such as genetic background and aging affect the single-cell immune landscape of influenza infection. Cytometry by time-of-flight (CyTOF) represents a promising technique in this pursuit, but interpreting its large, high-dimensional data remains difficult. We’ve developed a new analytical approach iGATE (in-silico gating annotating training elucidating) based on probabilistic support vector machine classification. By rapidly and accurately “gating” tens of millions of cells in silico into user-defined types, iGATE enabled us to track 25 canonical immune cell types in mouse lung over the course of influenza infection. Applying iGATE to study effects of host genetic background, we show that the lower survival of C57BL/6 mice compared to BALB/c is associated with a more rapid accumulation of inflammatory cell types and decreased IL-10 expression. Further, we demonstrate that the most prominent effect of aging is a defective T-cell response, reducing survival of aged mice. Finally, iGATE reveals that the 25 canonical immune cell types exhibit differential influenza infection susceptibility and replication permissiveness in vivo, but neither property varies with host genotype or aging. Software is available at https://github.com/UmichWenLab/iGATE.

Authors

Brett D. Hill, Andrew J. Zak, Sanjeev Raja, Luke F. Bugada, Syed M. Rizvi, Saiful B. Roslan, Hong Nhi Nguyen, Judy Chen, Hui Jiang, Akira Ono, Daniel R. Goldstein, Fei Wen