The periodic emergence of SARS-CoV-2 variants of concern (VOCs) with unpredictable clinical severity and ability to escape preexisting immunity emphasizes the continued need for antiviral interventions. Two small molecule inhibitors, molnupiravir (MK-4482), a nucleoside analog, and nirmatrelvir (PF-07321332), a 3C-like protease inhibitor, have recently been approved as monotherapy for use in high risk COVID-19 patients. As preclinical data are only available for rodent and ferret models, we here assessed the efficacy of MK-4482 and PF-07321332 alone and in combination against infection with the SARS-CoV-2 Delta VOC in the rhesus macaque COVID-19 model. Macaques were infected with the SARS-CoV-2 Delta variant and treated with either vehicle, MK-4482, PF-07321332 or a combination of MK-4482 and PF-07321332. Clinical exams were performed at 1, 2 and 4dpi to assess disease and virological parameters. Notably, use of MK-4482 and PF-07321332 in combination improved the individual inhibitory effect of both drugs resulting in milder disease progression, stronger reduction of virus shedding from mucosal tissues of the upper respiratory tract, stronger reduction of viral replication in the lower respiratory tract, and reduced lung pathology. Our data strongly indicate superiority of combined MK-4482 and PF-07321332 treatment of SARS-CoV-2 infections as demonstrated in the closest COVID-19 surrogate model.
Kyle Rosenke, Matthew C. Lewis, Friederike Feldmann, Eric Bohrnsen, Benjamin Schwarz, Atsushi Okumura, W. Forrest Bohler, Julie Callison, Carl Shaia, Catharine Bosio, Jamie Lovaglio, Greg Saturday, Michael Jarvis, Heinz Feldmann
Severe lung damage in COVID-19 involves complex interactions between diverse populations of immune and stromal cells. In this study, we used a spatial transcriptomics approach to delineate the cells, pathways and genes present across the spectrum of histopathological damage in COVID-19 lung tissue. We applied correlation network-based approaches to deconvolve gene expression data from 46 areas of interest covering >62,000 cells within well preserved lung samples from three patients. Despite substantial inter-patient heterogeneity, we discovered evidence for a common immune cell signaling circuit in areas of severe tissue that involves crosstalk between cytotoxic lymphocytes and pro-inflammatory macrophages. Expression of IFNG by cytotoxic lymphocytes was associated with induction of chemokines including CXCL9, CXCL10 and CXCL11 which are known to promote the recruitment of CXCR3+ immune cells. The tumour necrosis factor (TNF) superfamily members BAFF (TNFSF13B) and TRAIL (TNFSF10) were found to be consistently upregulated in the areas with severe tissue damage. We used published spatial and single cell SARS-CoV-2 datasets to confirm our findings in the lung tissue from additional cohorts of COVID-19 patients. The resulting model of severe COVID-19 immune-mediated tissue pathology may inform future therapeutic strategies.
Amy R. Cross, Carlos E. de Andrea, María Villalba-Esparza, Manuel F. Landecho, Lucia Cerundolo, Praveen Weeratunga, Rachel E. Etherington, Laura Denney, Graham Ogg, Ling-Pei Ho, Ian S.D. Roberts, Joanna Hester, Paul Klenerman, Ignacio Melero, Stephen N. Sansom, Fadi Issa
Understanding persistence and evolution of B cell clones after COVID-19 infection and vaccination is crucial for predicting responses against emerging viral variants and optimizing vaccines. Here, we collected longitudinal samples from severe COVID-19 patients every third to seventh day during hospitalization and every third month after recovery. We profiled the antigen-specific immune cell dynamics by combining single cell RNA-Seq, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE)-Seq, B cell receptor (BCR)-Seq with oligo-tagged antigen baits. While the proportion of Spike Receptor Binding Domain-specific memory B cells (MBC) increased from 3 months after infection, the other Spike- and Nucleocapsid-specific B cells remained constant. All patients showed ongoing class switching and sustained affinity maturation of antigen specific cells, which was not significantly increased early after vaccine. B cell analysis revealed a polyclonal response with limited clonal expansion; nevertheless, some clones detected during hospitalization, as plasmablasts, persisted for up to one year, as MBC. Monoclonal antibodies derived from persistent B cell families increased their binding and neutralization breadth and started recognizing viral variants by 3 months after infection. Overall, our findings provide important insights into the clonal evolution and dynamics of antigen specific B cell responses in longitudinally sampled COVID-19 infected patients.
Lydia Scharf, Hannes Axelsson, Aikaterini Emmanouilidi, Nimitha R. Mathew, Daniel J. Sheward, Susannah Leach, Pauline Isakson, Ilya V. Smirnov, Emelie Marklund, Nicolae Miron, Lars-Magnus Andersson, Magnus Gisslén, Ben Murrell, Anna Lundgren, Mats Bemark, Davide Angeletti
An animal model that fully recapitulates severe COVID-19 presentation in humans has been a top priority since the discovery of SARS-CoV-2 in 2019. Although multiple animal models are available for mild to moderate clinical disease, models that develop severe disease are still needed. Mink experimentally infected with SARS-CoV-2 developed severe acute respiratory disease, as evident by clinical respiratory disease, radiological, and histological changes. Virus was detected in nasal, oral, rectal, and fur swabs. Deep sequencing of SARS-CoV-2 from oral swabs and lung tissue samples showed repeated enrichment for a mutation in the gene encoding nonstructural protein 6 in open reading frame 1ab. Together, these data indicate that American mink develop clinical features characteristic of severe COVID-19 and, as such, are uniquely suited to test viral countermeasures.
Danielle R. Adney, Jamie Lovaglio, Jonathan E. Schulz, Claude Kwe Yinda, Victoria A. Avanzato, Elaine Haddock, Julia R. Port, Myndi G. Holbrook, Patrick W. Hanley, Greg Saturday, Dana Scott, Carl Shaia, Andrew M. Nelson, Jessica R. Spengler, Cassandra Tansey, Caitlin M. Cossaboom, Natalie M. Wendling, Craig Martens, John Easley, Seng Wai Yap, Stephanie N. Seifert, Vincent J. Munster
Loss of olfactory function has been commonly reported in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections. Recovery from anosmia is not well understood. Previous studies showed that sustentacular cells, and occasionally, olfactory sensory neurons (OSNs) in the olfactory epithelium (OE) are infected in SARS-CoV-2-infected patients and experimental animals. Here, we show that SARS-CoV-2 infection of sustentacular cells induces inflammation characterized by infiltration of myeloid cells to the olfactory epithelium and variably increased expression of proinflammatory cytokines. We observed widespread damage to, and loss of cilia on, OSNs, accompanied by downregulation of olfactory receptors and signal transduction molecules involved in olfaction. A consequence of OSN dysfunction was a reduction in the number of neurons in the olfactory bulb expressing tyrosine hydroxylase, consistent with reduced synaptic input. Resolution of the infection, inflammation, and olfactory dysfunction occurred over 3-4 weeks following infection in most but not all animals. We also observed similar patterns of OE infection and anosmia/hyposmia in mice infected with other human coronaviruses such as SARS-CoV and MERS-CoV. Together, these results define the downstream effects of sustentacular cell infection and provide insight into olfactory dysfunction in COVID-19-associated anosmia.
Abhishek Kumar Verma, Jian Zheng, David K. Meyerholz, Stanley Perlman
Despite the widespread use of SARS-CoV-2-specific monoclonal antibody (mAb) therapy for the treatment of acute COVID-19, the impact of this therapy on the development of SARS-CoV-2-specific T cell responses has been unknown, resulting in uncertainty as to whether anti-SARS-CoV-2 mAb administration may result in failure to generate immune memory. Alternatively, it has been suggested that SARS-CoV-2-specific mAb may enhance adaptive immunity to SARS-CoV-2 via a "vaccinal effect." Bamlanivimab (Eli Lilly) is a recombinant human IgG1 that was granted FDA emergency use authorization for the treatment of mild to moderate COVID-19 in those at high risk for progression to severe disease. Here, we compared SARS-CoV-2 specific CD4+ and CD8+ T cell responses of 95 individuals from the ACTIV-2/A5401 clinical trial 28 days after treatment with 700 mg bamlanivimab versus placebo. SARS-CoV-2-specific T cell responses were evaluated using activation induced marker (AIM) assays in conjunction with intracellular cytokine staining. We demonstrate that most individuals with acute COVID-19 develop SARS-CoV-2-specific T cell responses. Overall, our findings suggest that the quantity and quality of SARS-CoV-2-specific T cell memory was robust in individuals who received bamlanivimab for acute COVID-19. Receipt of bamlanivimab during acute COVID-19 neither diminished nor enhanced SARS-CoV-2-specific cellular immunity.
Sydney I. Ramirez, Alba Grifoni, Daniela Weiskopf, Urvi M. Parikh, Amy Heaps, Farhoud Faraji, Scott F. Sieg, Justin Ritz, Carlee B. Moser, Joseph J. Eron, Judith S. Currier, Paul Klekotka, Alessandro Sette, David A. Wohl, Eric S. Daar, Michael D. Hughes, Kara W. Chew, Davey M. Smith, Shane Crotty
Consecutive mRNA vaccinations against SARS-CoV-2 reinforced both innate and adaptive immune responses. However, it remains unclear whether the enhanced innate immune responses are mediated by epigenetic regulation and, if so, whether these effects persist. Using mass cytometry, RNA-seq, and ATAC-seq, we show that BNT162b2 mRNA vaccination upregulated antiviral and IFN-stimulated gene expression in monocytes with greater effects after the second vaccination than those after the first vaccination. Transcription factor-binding motif analysis also revealed enriched IFN regulatory factors and PU.1 motifs in accessible chromatin regions. Importantly, although consecutive BNT162b2 mRNA vaccinations boosted innate immune responses and caused epigenetic changes in isolated monocytes, we showed that these effects occur only transiently and disappear 4 weeks after the second vaccination. Furthermore, single-cell RNA sequencing analysis revealed that a similar gene signature was impaired in the monocytes of unvaccinated COVID-19 patients with acute respiratory distress syndrome. These results reinforce the importance of the innate immune response in the determination of COVID-19 severity but indicate that, unlike adaptive immunity, innate immunity is not unexpectedly sustained even after consecutive vaccination. This study, which focuses on innate immmune memory, may provide novel insights into the vaccine development against infectious diseases.
Yuta Yamaguchi, Yasuhiro Kato, Ryuya Edahiro, Jonas N. Søndergaard, Teruaki Murakami, Saori Amiya, Shinichiro Nameki, Yuko Yoshimine, Takayoshi Morita, Yusuke Takeshima, Shuhei Sakakibara, Yoko Naito, Daisuke Motooka, Yu-Chen Liu, Yuya Shirai, Yasutaka Okita, Jun Fujimoto, Haruhiko Hirata, Yoshito Takeda, James B. Wing, Daisuke Okuzaki, Yukinori Okada, Atsushi Kumanogoh
Protective immunity against SARS-CoV-2 infection after COVID-19 vaccination may differ by variant. We enrolled vaccinated (n = 39) and unvaccinated (n = 11) individuals with acute, symptomatic SARS-CoV-2 Delta or Omicron infection and performed SARS-CoV-2 viral load quantification, whole-genome sequencing, and variant-specific antibody characterization at the time of acute illness and convalescence. Viral load at the time of infection was inversely correlated with antibody binding and neutralizing antibody responses. Across all variants tested, convalescent neutralization titers in unvaccinated individuals were markedly lower than in vaccinated individuals. Increases in antibody titers and neutralizing activity occurred at convalescence in a variant-specific manner. For example, among individuals infected with the Delta variant, neutralizing antibody responses were weakest against BA.2, whereas infection with Omicron BA.1 variant generated a broader response against all tested variants, including BA.2.
Michael S. Seaman, Mark J. Siedner, Julie Boucau, Christy L. Lavine, Fadi Ghantous, May Y. Liew, Josh I. Mathews, Arshdeep Singh, Caitlin Marino, James Regan, Rockib Uddin, Manish C. Choudhary, James P. Flynn, Geoffrey Chen, Ashley M. Stuckwisch, Taryn Lipiner, Autumn Kittilson, Meghan Melberg, Rebecca F. Gilbert, Zahra Reynolds, Surabhi L. Iyer, Grace C. Chamberlin, Tammy D. Vyas, Jatin M. Vyas, Marcia B. Goldberg, Jeremy Luban, Jonathan Z. Li, Amy K. Barczak, Jacob E. Lemieux
Cross-reactive immunity between SARS-CoV-2 and other related coronaviruses has been well-documented, and it may play a role in preventing severe COVID-19. Epidemiological studies early in the pandemic showed a geographical association between high influenza vaccination rates and lower incidence of SARS-CoV-2 infection. We, therefore, analyzed whether exposure to influenza A virus (IAV) antigens could influence the T cell repertoire in response to SARS-CoV-2, indicating a heterologous immune response between these 2 unrelated viruses. Using artificial antigen-presenting cells (aAPCs) combined with real-time reverse-transcription PCR (RT-qPCR), we developed a sensitive assay to quickly screen for antigen-specific T cell responses and detected a significant correlation between responses to SARS-CoV-2 epitopes and IAV dominant epitope (M158–66). Further analysis showed that some COVID-19 convalescent donors exhibited both T cell receptor (TCR) specificity and functional cytokine responses to multiple SARS-CoV-2 epitopes and M158–66. Utilizing an aAPC-based stimulation/expansion assay, we detected cross-reactive T cells with specificity to SARS-CoV-2 and IAV. In addition, TCR sequencing of the cross-reactive and IAV-specific T cells revealed similarities between the TCR repertoires of the two populations. These results indicate that heterologous immunity shaped by our exposure to other unrelated endemic viruses may affect our immune response to novel viruses such as SARS-CoV-2.
Worarat Chaisawangwong, Hanzhi Wang, Theodore Kouo, Sebastian F. Salathe, Ariel Isser, Joan Glick Bieler, Maya L. Zhang, Natalie K. Livingston, Shuyi Li, Joseph J. Horowitz, Ron E. Samet, Israel Zyskind, Avi Z. Rosenberg, Jonathan P. Schneck
People living with HIV-1 (PLWH) exhibit more rapid antibody decline following routine immunization and elevated baseline chronic inflammation than people without HIV-1 (PWOH), indicating potential for diminished humoral immunity during SARS-CoV-2 infection. Conflicting reports have emerged on the ability of PLWH to maintain humoral protection against SARS-CoV-2 co-infection during convalescence. It is unknown if peak COVID-19 severity, along with HIV-1 infection status, associates with the quality and quantity of humoral immunity following recovery. Using a cross-sectional observational cohort from the USA and Peru, adults were enrolled 1-10 weeks post-SARS-CoV-2 infection diagnosis or symptom resolution. Serum antibodies were analyzed for SARS-CoV-2-specific response rates, binding magnitudes, ACE2 receptor blocking and antibody dependent cellular phagocytosis (ADCP). Overall, (1) PLWH exhibited a trend towards decreased magnitude of SARS-CoV-2-specific antibodies, despite modestly increased overall response rates when compared to PWOH, (2) PLWH recovered from symptomatic outpatient COVID-19 had comparatively diminished immune responses, and (3) PLWH lacked a corresponding increase in SARS-CoV-2 antibodies with increased COVID-19 severity when comparing asymptomatic to symptomatic outpatient disease.
Daniel J. Schuster, Shelly Karuna, Caroline Brackett, Martina S. Wesley, Shuying S. Li, Nathan Eisel, DeAnna Tenney, Sir'Tauria Hilliard, Nicole L. Yates, Jack R. Heptinstall, LaTonya D. Williams, Xiaoying Shen, Robert Rolfe, Robinson Cabello, Lu Zhang, Sheetal Sawant, Jiani Hu, April Kaur Randhawa, Ollivier Hyrien, John A. Hural, Lawrence Corey, Ian Frank, Georgia D. Tomaras, Kelly E. Seaton
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