bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of swine Fc conjugated Serotype O Foot and mouth disease virus like particles
Authors: Shin Hyun-Jin , Park Jae-Yeon
Published: January 2023

Foot-and mouth disease virus (FMDV) cause a highly contagious infection in cloven-hoofed animals. The current conventional FMD vaccines are produced by chemical inactivated virulent FMDV virions, but conventional FMD vaccines risky and limitation such as respect to accidental release of FMDV and high-level biosafety facilities were required to handle FDMV. FMDV virus like particle (VLP) could be alternative choice for future vaccine against FMDV because of efficacy and safety. Receptor for Fc portion of antibody play and important role in the activation of immune response for infection of virus and bacteria. Therefore, immunization with a complex of virus antigen and Fc has the potential to be an improved. Here we report our vaccine studies with Fc conjugated serotype O FMDV VLP for alternative FMDV vaccine.
Identifying B cells in hatchling and adult Trachemys scripta, red-eared slider turtles
Authors: Paton Hanna D , Mool Allison , Fairow Christen , Hurst Whitney , Bowden Rachel , Vogel Laura
Published: January 2023

Evidence suggests there are important differences between reptile and mammalian immune strategies, yet little is known about humoral immune responses in reptilian vertebrates. Our laboratory is interested in reptile B cell development and function and our undergraduate research project involved B cell detection using flow cytometry in spleens of hatchling and adult turtles. We made use of a previously developed monoclonal antibody (HL673) that recognizes turtle light chain protein. The murine HL673 mAb was purified using a protein A affinity column and reactivity was tested by ELISA. Some of the purified antibody was biotinylated and incubated with blood and spleen samples from adult and hatchlings. Bound antibodies were detected and B cell populations identified using flow cytometry. We were able to identify B cell containing populations based on scatter and specific staining. Our results showed detection of turtle B cells using the labeled mAb in both adults and 6 week old hatchlings. Future studies will use this reagent to further investigate the distribution and function of B cells.

This work was supported by NSF 1725199 and NIH 1R15AI140118 – 01.
Flt3 and its ligand as an ancient regulators of dendritic cells: evidence in the amphibian Xenopus laevis
Authors: Paiola Matthieu , Roy Sobhan , Ma Siyuan , Patrick Charkira , Pavelka Marin , Adams Erin J , Robert Jacques
Published: January 2023

The evolution of hematopoietic-derived dendritic cells that are so critical for T cell activation is still poorely understood. In mammals, fms related tyrosine kinase 3 (Flt3) and its ligand (Flt3lg) are important regulator of dendritic cell homeostasis. Flt3 and Flt3lg co-evolution was investigated in all jawed vertebrates with special focus in an ancient allotetraploid tetrapod Xenopus laevis. flt3 synteny and predicted amino-acid structure are conserved in all jawed vertebrate including in the duplicated S and L homeologous chromosomes of X. laevis. Flt3lg gene is conserved among X. laevis homeologous chromosomes and most jaw vertebrates with the exception of ray-finned fish. Amino acid comparison, 3D structure modelling and tissue distribution suggest a subfunctionalization of Flt3 and Flt3lg homeologs. Tagged recombinant Flt3lg.S and Flt3lg.L were produced to investigate cell surface expression of Flt3s. Both Flt3lg.S and L specifically stain a splenic population of leukocyte MHC class IIhigh+ and CD8+ phenotypically similar to the recently described X. laevis dual follicular/dendritic-like XL cells. Our results indicate that Flt3lg.S and L are both involved in XL cell homeostasis and that XL cells have hematopoietic origin. In X. laevis, the non-polymorphic MHC class I-like XNC4 restricting innate-like T cells is critical for tadpoles’ resistance against the non-TB mycobacteria M.marinum. Preliminary experiments indicate that XL cells are XNC4+ and are being recruited to the infection site together with XNC4-restricted T cells. This suggests that XL cells are involved in T cell activation, which is consistent with the an ancestral dual follicular/dendritic cell function of XL cells.

Supported by NIH (R24-AI059830 – R21-AI139718), NSF (1754274) and 2021 Rochester Vaccine Fellowship
The human IGHD3-22 encoded motif contributes to broad reactivity of anti-SARS-CoV-2 antibodies
Authors: Liu Hejun , Wilson Ian A
Published: January 2023

The continuous evolution of SARS-CoV-2 coronavirus, the etiological agent of COVID-19, has resulted in the accumulation of mutations that may either escape from host immunity or increase its transmissibility. Cross-neutralizing antibodies targeting highly conserved sites of sarbecoviruses are critical to combat these SARS-CoV-2 variants and other related sarbecoviruses. However, these antibodies are apparently less frequently isolated, although robust antibody responses have been observed in many COVID-19 patients and vaccinees. Here, we describe a structural motif encoded by the human IGHD3-22 gene that targets a functionally conserved surface on the spike protein across sarbecoviruses. We compared antibody structures binding to this conserved site and found a recurrent YYDRxG motif within cross-neutralizing antibodies that mediates strong polar and hydrophobic interactions to the receptor-binding domain of SARS-CoV-2. A computational search using the YYDRxG pattern with publicly available antibody sequences yield over 100 such antibodies with many that broadly neutralize SARS-CoV-2 variants of concern, as well as other sarbecoviruses. Our analysis revealed a common convergent solution to counteract sarbecoviruses, but which seems to be elicited less frequently than other responses by the human humoral immune system. Nevertheless, statistical analysis shows that these YYDRxG sequences are commonly elicited in COVID-19 patients and vaccinees, providing a promising strategy that could be implemented in broad anti-sarbecovirus antibody identification and for pan-sarbecovirus vaccine development.
SARS-CoV-2 vaccination induces T cell memory responses able to cross-recognize ongoing SARS-CoV-2 variants including omicron.
Authors: Grifoni Alba , Tarke Alison , Dan Jennifer , Coelho Camila , Zhang Zeli , Weiskopf Daniela , Da Silva Antunes Ricardo , Crotty Shane , Sette Alessandro
Published: January 2023

The emergence of SARS-CoV-2 variants raises concerns about the efficacy of COVID-19 vaccines to induce an immune response capable of recognizing upcoming variants. While there are multiple studies detailing the impact of SARS-CoV-2 variants on neutralizing antibodies, there is relatively little information on the effect of variant mutations on CD4 and CD8 T cell recognition. We will present the results of our ongoing study on the effects on T cell reactivity to a comprehensive panel of variants circulating globally (B.1.617.2 [delta], P.1 [gamma], B.1.617.1 [kappa], C.37 [lambda], R.1, B.1.1.7 [alpha], B.1.351 [beta], B.1.1.519, B.1.525 [eta], B.1.526 [iota], B.1.427/9 [epsilon], B.1.526.1 and B.1.621 [mu] comparing it with the most recent B.1.1.529 [omicron] variant. The results will span from bioinformatics analyses addressing the effect of SARS-CoV-2 mutations of known epitopes in the Immune Epitope Database (IEDB, https://www.iedb.org/) to the experimental evaluation of the T cell responses specific to each SARS-CoV-2 Spike variant in individuals immunized with several vaccine platforms and at multiple timepoints after vaccination. Parallel experiments will also determine the epitope repertoire in specific donors and dissect the impact of SARS-CoV-2 variant associated mutations on the recognition of each CD4 and CD8 T cell epitope. Finally, the capability of T cell cross-recognition will be compared with breakthrough infection and with or without booster vaccination.

This project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. 75N93021C00016 to A.S. and Contract No. 75N9301900065 to A.S, D.W.
Galectin-9 protects humanized-ACE2 immunocompetent mice from SARS-CoV-2 Infection
Authors: Yeung Stephen T , Premeaux Thomas A , Niki Toshiro , Pillai Satish K , Khanna Kamal M , Ndhlovu Lishomwa C
Published: January 2023

The COVID19 pandemic caused by SARS CoV 2 remains a global health crisis even with vaccines and sparse FDA approved therapies. To limit mortality and morbidity, therapeutics are critical. Galectin 9 (gal9) is a b-galactoside binding protein, modulating cell-cell and cell-matrix interactions. It has been shown circulating gal9 levels are elevated in response to infection in patient sera with moderate to severe disease. One report gal9 unexpectedly competitively binds the ACE2 receptor on host cells, impeding viral entry. Therefore, we hypothesize early recombinant gal9 (rgal9) treatment post infection (pi) may prevent binding of the virus to prone host cells yielding a decreased severity of SARS CoV 2 related disease. To determine the potential of gal9 for treating COVID19, we infected and treated K18 hACE2 mice with a single dose of human rgal9 (rhgal9) or PBS i.p. and monitored 12 days for morbidity. Subgroups of mice were humanely euthanized at 2 and 5 dpi for viral plaque assay, flow cytometry, and protein analysis from lung tissue and BALF. We found that mice treated with rhgal9 during acute phase of infection have improved survival compared to PBS control. At 5 dpi, rhgal9 mice had enhanced viral clearance in the BALF and not in the lung parenchyma. We noted increased CD8 T cell and decreased neutrophil frequencies in the lung at 5 dpi. Finally, BALF had elevated levels of Type 1 IFN at 2 dpi and increased proinflammatory cytokines at 5 dpi. This suggests rhgal9 treatment may be therapeutic for acute COVID19, as rhgal9 with other anti-inflammatory mediator treatments cut associated harmful inflammation. Further studies are required to determine the optimal time, combination and duration of treatment pi to effectively target the gal9 pathway.
Endogenous interferon-lambda signaling restricts virus replication and disease severity in a murine model of SARS-CoV-2 infection
Authors: Solstad Abigail , Kenney Adam D , Zani Ashley , Yount Jacob S , Hemann Emily A
Published: January 2023

Interferon-lambda (IFN-λ) is currently being investigated in a phase III clinical trial as a therapeutic against COVID-19. Exogenous IFN-λ restricts SARS-CoV-2 in vitro and in Balb/c and C57Bl/6 (WT) murine models of infection. However, roles of endogenously produced IFN-λ in SARS-CoV-2 pathogenesis are not currently known, and the overall mechanisms by which IFN-λ modulates the induction of protective immune responses in SARS-CoV-2 infections remains to be elucidated. We find that IFN-λ receptor deficient mice (Ifnlr1−/−) infected with mouse-adapted SARS-CoV-2 lose significantly more weight and have increased SARS-CoV-2 viral replication compared to WT through day 5 post infection. Intriguingly, Ifit3 and Ifitm3 are increased in the lungs of Ifnlr1−/− mice compared to WT following infection, despite similar IFN-α/β and IFN-λ mRNA levels, suggesting compensatory increases in type I IFN signaling are not driving the increased weight loss or ISG induction observed in Ifnlr1−/− mice. Global transcriptomics revealed induction of a suppressive immunoregulatory signature with increased IL-10 as a hallmark in Ifnlr1−/− lungs, suggesting IFN-λ is critically involved in regulating appropriate immune activation to limit SARS-CoV-2 pathogenesis. Histological analysis revealed a significant increase in CD45+ cells (but not neutrophils) in the lungs of Ifnlr1−/− mice compared to WT on day 5 post infection, and we identified increases in pathways associated with myeloid cell function and T cell activation in Ifnlr1−/− by comparative transcriptomics. Overall, broadening the understanding of how IFN-λ regulates SARS-CoV-2 infection, pathogenesis, and immunity will inform the utilization of IFN-λ as an immunotherapy and adjuvant.

Supported by NIH Award K22 AI146141 to EAH.
Analysis of SARS-CoV-2 pathogenicity in COVID-19 cynomolgus macaque model reflecting human COVID-19 pathological conditions
Authors: Urano Emiko , Okamura Tomotaka , Yasutomi Yasuhiro
Published: January 2023

The pandemic of SARS-CoV-2 is still raging worldwide. Although rapid vaccination has been progressing in the world as a countermeasure against COVID-19, there are many issues regarding this disease including the emergence of variants and of their virulence that need to be elucidated, and a useful animal model is still necessary.

We have established COVID-19 cynomolgus macaque (CM) model using healthy young CMs and CMs of advanced age (23 – 30 years of age, equivalent to 69 – 90 years of age in humans) with underlying diseases including diabetes and hyperlipidemia. There were differences between the pathological conditions in young macaques and elderly macaques of advanced age similar to observed in humans. In this study, we analyzed the virulence and pathology of SARS-CoV-2 variants of concern (VOCs) using COVID-19 CM model.

Elucidation of how adaptive immunity in patients is induced by SARS-CoV-2 natural infection is critical for establishing a vaccine strategy, serologic therapies, public health control and prediction of infection. Therefore, we conducted the prolonged rechallenge analysis using COVID-19 CM model reinoculated with either the same or a different strain of SARS-CoV-2 that was used for the initial infection. Protective immunity against rechallenge of SARS-CoV-2 is under consideration, but in one elderly monkey reinfection showed transient pneumonia with increased levels of various serum cytokines and chemokines without detecting viruses in swab samples.

Overall, the SARS-CoV-2-infected CM models reflected the pathology in humans, and studies using COVID-19 CM models should contribute to the elucidation of the complicated pathophysiology of COVID-19.

Supported by grants from AMED (JP20fk0108414) and JSPS (21K19395)
Immune responses to SARS-Cov-2 variants in adult and elderly mRNA vaccine recipients
Authors: Jergovic Mladen , Uhrlaub Jennifer , Watanabe Makiko , Coplen Christopher P , Nikolich-Zugich Janko
Published: January 2023

Aging is associated with a reduced magnitude of primary immune responses to vaccination and constriction of immune receptor repertoire diversity. Clinical trials demonstrate high efficacy of mRNA based SARS-CoV-2 vaccines in the elderly but concerns about virus variant escape have not been well addressed. Recently, we have conducted an in-depth analysis of humoral and cellular immunity against the early-pandemic virus strain as well as the P.1./Gamma, B.1.617/Delta and B.1.595 SARS-CoV-2 variants in adult and elderly mRNA vaccine recipients. As others have reported, robust immunity required the second dose of vaccine. Older vaccine recipients exhibited an expected 3–5x reduction (but not a complete loss) in neutralizing antibody titers against P.1./Gamma and B.1.595 that did not statistically differ from that measured in adults. Moreover, older vaccinees manifest robust cellular immunity against SARS-CoV-2, including to variants, that remained statistically comparable to the adult group. While the duration of these immune responses remains to be determined over longer periods of time, these results provide reasons for optimism regarding vaccine protection of older adults against SARS-CoV-2 variants. Experiments are currently in progress to determine cellular immunity in adult and older adult mRNA vaccine recipients to Omicron (B.1.1.529), the newest variant of concern. We will present results on T cell polyfunction and production of cytokines in response to Spike protein peptide pool from Omicron variant in a large cohort of convalescents and adult and elderly mRNA vaccine recipients.
Repurposed antibiotic and antiviral drugs inhibit the immune evasive endoribonuclease of SARS-CoV-2 and restrict coronavirus infection in vitro
Authors: von Beck Troy Alexander , Hernandez Luis Mena , Zhou Hongyi , Skolnick Jeffrey , Jacob Joshy
Published: January 2023

Despite the success of SARS-CoV-2 vaccines in curbing viral transmission and severe disease, infections in unvaccinated and immune deficient patients continue to drive significant morbidity and mortality globally. To address the needs of this patient population, multiple antiviral drugs must be developed to expand on the protection currently offered by convalescent plasma, monoclonal antibodies, remdesivir, and other next generation SARS-CoV-2 antivirals which may be contraindicated for some patient groups. To this end, we employed a machine-learning based virtual ligand screening algorithm, FRAGSITE, to screen a library of FDA approved compounds for binding to the SARS-CoV-2 endoribonuclease (nsp15), a structurally conserved and demonstrated coronavirus virulence factor in SARS-CoV-1, HCoV-229E, IBV, MHV, and PEDV. Using recombinant SARS-CoV-2 nsp15, we identified the inhibition of nuclease activity by 11 drugs in vitro; 6 also restrict infection by the related human OC43 coronavirus in human A549 airway epithelial cells as measured by a focus forming assay. Among these 6 candidates, pibrentasvir remained active against HCoV-OC43 at the lowest concentration (IC50 < 0.625μM). Consistent with previous findings, HCoV-OC43 infection of A549 cells did not provoke an innate response to the viral dsRNA as quantified by IFNB1 mRNA production, antiviral stress granule formation, or RNase L activation. Our preliminary results suggest that antiviral stress granule formation is partially restored by nsp15 inhibitor treatment in HCoV-OC43 infected cells, although IFNβ production and RNase L activation remain unchanged.

Supported by grants from the Emory School of Medicine
SARS-CoV-2 epitope-specific CD8+ clonotypes persist 8 month post-infection despite reduced clonality.
Authors: Zornikova Ksenia V , Khmelevskaya Alexandra , Sheetikov Savely , Shcherbakova Olga V , Kirukhin Dmitry , Titov Aleksei , Zvyagin Ivan V , Efimov Grigory A
Published: January 2023

Memory T cells play a crucial role in acceleration of immune response during reinfection with the same pathogen. Since the beginning of the COVID-19 pandemic, attention has been focused on determining the epitopes of SARS-CoV-2 that give rise to long-lived memory T cells.

We examined the immune response to 12 immunogenic CD8+ epitopes in 26 COVID-19 convalescent patients. Paired blood samples were collected shortly after infection and approximately 8 months after. Using ex vivo T cell expansions with a subsequent MHC-tetramer+ cell-sorting and T cell receptor beta-chain (TCRβ) sequencing we studied the epitope-specific CD8+ response on the clonal level. In total in both time points we identified 756 epitope-specific TCRβ sequences. Nine epitopes were immunodominant while five immunogenic epitopes (YLQ, KCY, LLY, KTF and ALW) yielded response in 100% of tested individuals. This coincided with a high number of clones per individual specific to these epitopes (median = 16.5).

Despite the very low frequency of specific cells in the total repertoire most (75.4%) of responses were retained at 8-month post-infection. The median number of clones for the top five most immunogenic epitopes dropped to 6. The TCRβ repertoires of T cells specific to the studied epitopes were characterized by different degrees of CDR3 homology. LLY and YLQ-specific TCRβ had the highest degree of mutual similarity while KCY and KTF-specific TCRβ were the most diverse.

Our data demonstrates that despite the overall decrease of T cell reactivity and clonality most epitopes are still recognized due to the ability of memory SARS-CoV-2-specific CD8+ cells to survive over prolonged periods of time.

The work was supported by the Russian Science Foundation grant 20-15-00395.
A Longitudinal Study of Humoral Immune Responses to SARS-CoV-2 Spike Proteins in Gamma-Interferon-inducible Lysosomal Thiol Reductase Deficient Mice
Authors: He Xiao , Guo Li , Dai Hu , Jensen Peter E
Published: January 2023

The disulfide (S-S) bonds located in the receptor binding domain (RBD) of Spike (S) proteins of SARS-CoV-2 are critical for structural folding and viral entry. Once entered cells, S proteins, like other exogenous Ag, can be transported into endo/lysosomes, where gamma-interferon-inducible lysosomal thiol reductase (GILT) is the only thiol reductase located, which can reduce S-S bonds. GILT mediated S-S bond reduction in APC is critical in unfolding of native S-S bonds containing Ag, subsequently processing/presenting Ag, and, finally, triggering CD4 T cell responses. Recently, we also find GILT plays a role in Ab responses. Whether GILT mediates anti-SARSCoV-2 host defense responses remains unknown. We hypothesized that GILT is important in eliciting host humoral immune responses against SARS-CoV-2 by reducing S-S bonds and exposing Ag epitopes. To address this, we used 3 different types of S protein derived Ag to immunize GILT KO or B6 control mice and measured the specific Ab responses in a longitudinal study. GILT KO or B6 wildtype mice were given RBD, S1 region and the full length of extracellular domain of S proteins with CFA initially, and boosted at week 3 with the same Ag in IFA. Sera were collected at weeks 3 and 5, months 3, 4 and 5 for quantification of specific anti-RBD, S1 and S Ab. At all timepoints examined, GILT KO mice generated lower specific Ab titers to the immunogens, as compared to B6. Furthermore, decay of anti-specific Ab in GILT KO was faster than that in controls after immunization. Our preliminary findings showed an important role of GILT in humoral immune responses against SARS-CoV-2, suggesting unfolding S-S bonds is critical in this process. Recombinant GILT as a therapeutic might benefit patients with COVID-19.

Supported by Seed Grant, Univ. of Utah
Epitope-specific T cell response to SARS-CoV-2 infection and vaccination
Authors: Minervina Anastasia , Pogorelyy Mikhail , Kirk Allison M , Chou Ching-Heng , Allen E Kaitlynn , Crawford Jeremy Chase , McGargill Maureen Ann , Thomas Paul G
Published: January 2023

SARS-CoV-2 mRNA vaccines are effective for COVID-19 prevention, eliciting both robust antibody responses in naive individuals and boosting antibody levels in convalescent donors. However, the effect of repeated antigen exposures, such as vaccination following infection or breakthrough infections, on the magnitude, repertoire, and phenotype of pre-existing memory T cells, is still poorly understood. Thus, we compared epitope-specific CD8 T cells elicited after SARS-CoV-2 infection, vaccination of both naive and recovered individuals, and breakthrough infection cases. We used pools of 18 DNA-barcoded MHC-class I multimers, combined with scRNAseq and scTCRseq, to characterize T cell responses as defined by magnitude, specificity, T cell receptor (TCR) repertoire, and gene expression profile to both spike-derived and non-spike derived epitopes. In-depth analysis of over 4000 unique epitope-specific TCR sequences demonstrates that both vaccination and infection, including breakthrough cases, elicit identical repertoires as measured by dominant TCR motifs and repertoire diversity, indicating that BNT162b2 vaccination largely recapitulates spike-specific T cell repertoire generation by infection. Importantly, in COVID-19-recovered individuals receiving the vaccine, pre-existing spike-specific memory cells showed both clonal expansion and a phenotypic shift towards more differentiated CCR7-CD45RA+ effector cells, demonstrating the potency of this vaccine to recall spike-specific CD8 memory T cells. Importantly, in breakthrough infections, we observed a high proportion of T cells targeting non-spike epitopes, showing that new immune memory could be formed during SARS-CoV-2 infection after vaccination.

The work was funded by 75N93019C00052, HHSN272201400006C, 3U01AI144616-02S1, R01AI136514
Metabolic syndrome enhances viral disease severity and reduces vaccine efficacy in mice
Authors: Geerling Elizabeth , Carpenter Danielle H , Schwetye Katherine E , DeBosch Brian , Pinto Amelia
Published: January 2023

Metabolic syndrome (MetS) is a cluster of conditions linked by chronic inflammation that increase the risk for comorbidities. Currently, 1/3 of United States adults have MetS. Globally, obesity rates have tripled since 1975, which is notable since it is a criterium used to diagnose MetS. MetS patients show higher mortality post-SARS-CoV-2 infection and poor vaccination outcomes following influenza virus vaccination when compared to metabolically healthy humans. However, the mechanisms driving MetS-induced immune dysfunction are unknown. Based on previously published work, we hypothesize that MetS-associated chronic inflammation alters programming of adaptive immune cells critical for viral control, thus enhancing disease severity and reducing vaccine efficacy. To challenge this, we use West Nile virus and SARS-CoV-2 in MetS murine models. By infecting or vaccinating chow fed wild type (WT) and high fat diet induced MetS mice, we compared immune responses over time. Our results indicate that MetS mice have higher mortality post infection, heightened viral titers, severe organ pathology, dysfunctional T and B cell responses and reduced neutralizing antibody efficacy when compared to WT mice. Further, MetS alters antibody and T cell responses post-vaccination, rendering vaccination insufficient for protecting against severe viral disease. Our results imply that MetS enhances viral disease severity and reduces vaccine efficacy. Ongoing studies in our lab are focused on determining the mechanism by which MetS alters adaptive immune cell function, with our data implicating chronic inflammation as an inducer of immune cell epigenetic changes that alter their differentiation landscapes and consequently effector functions.

Supported by grants from NIH (R01 12781495) and DoD (USAMRDCPR192269).
ADAM-17 protease promotes inflammation and mortality while decreasing viral burden in a COVID-19 mouse model
Authors: Hedges Jodi F , Snyder Deann T , Robison Amanda , Walk Heather , Havlak Karlin , Shepardson Kelly , Kominsky Douglas , Rynda-Apple Agnieszka , Walcheck Bruce , Jutila Mark A
Published: January 2023

Angiotensin Converting Enzyme 2 (ACE2) is the primary cell entry receptor for SARS-CoV-1 and SARS-CoV-2 viruses. A disintegrin and metalloproteinase 17 (ADAM-17) is a protease located in the cell membrane of most cells that, upon cellular activation, cleaves ectodomains of transmembrane proteins, including that of ACE2, from cell surfaces. We hypothesized that blockade of ADAM-17 activity would alter COVID-19 pathogenesis. To assess this pathway, we blocked the function of ADAM-17 using a monoclonal antibody in the K18 human ACE2 Tg mouse model of COVID-19. Antibody-treated mice were healthier, less moribund, and had significantly less lung inflammation than saline treated mice. However, the viral burden in the lungs of anti-ADAM17 Ab-treated mice was significantly greater. ADAM17 also cleaves TNF-a and its blockade decreased lung TNF-a production induced by intratracheal LPS delivery. We are currently testing this possibility in the COVID-19 mouse model. Thus, ADAM-17 appears to have a critical anti-viral role, but also promotes damaging inflammation following SARS-CoV-2 infection. Since the inflammatory cascade is ultimately the reason for the adverse outcomes in COVID-19 patients, there may be a therapeutic application for the anti-ADAM-17 antibody.
Novel mouse model of MCMV-induced adaptive NK cells
Authors: Jensen Isaac J , Martin Matthew D , Tripathy Sandeep K , Badovinac Vladimir P , Farber Donna L
Published: January 2023

NK cells are important mediators of viral control with the capacity to form adaptive immune features following infection. However, studies of infection-induced adaptive NK cells require adoptive cell transfer to lower the precursor frequency of ‘antigen-specific’ NK cells, potentially limiting the diversity of the NK cell response. In seeking an unmanipulated model to probe the adaptive NK cells, we interrogated a wide range of Collaborative Cross (CC) inbred mice, inbred mouse strains which exhibit broad genetic diversity across strains. Our assessment identified and validated a putative ‘ideal’ CC strain, CC006, which does not require manipulation to generate and maintain adaptive NK cells. Critically, CC006 mice, in contrast to C57Bl/6 mice, are capable of developing enhanced NK cell-mediated protective responses to MCMV infection following m157-mediated vaccination. This work both furthers our understanding of adaptive NK cells and demonstrates the utility of CC mice in the development and interrogation of immunologic models.

Supported by T32AI007511 (IJJ), T32AI007485 (IJJ), 5R01AI114543 (VPB), 1R35GM134880 (VPB), U01-AI150680 (DLF)
Liver Biopsies Reveal Temporal Changes in Pathology and Macrophage Function following SIV Infection of Rhesus Macaques
Authors: Derby Nina R , Fancher Katherine A , Biswas Sreya , Yusova Sofiya , Luevano-Santos Cristina , Smedley Jeremy , Pacheco Cristina , Burwitz Benjamin , Sodora Donald L
Published: January 2023

Non-alcoholic fatty liver disease (NAFLD) contributes to morbidity and mortality in HIV-infected individuals. To evaluate liver immune changes during SIV infection of macaques, we obtained liver biopsies laparoscopically at 2, 6, and 16 weeks post-infection as well as at necropsy (32 weeks). SIV infection was associated with liver dysfunction that included elevated liver enzymes during acute infection, higher liver weight at necropsy, and histologic changes within the liver lobule (functional unit of the liver) including sinusoidal dilatation near central veins and portal expansion. The type-1 interferon (IFN-1) response (MX1 expression) increased over time post-SIV infection, initially being observed in macrophages (week 2) with a switch to a mixed hepatocyte/monocyte/macrophage response by 16–32 weeks. Over the course of SIV infection, macrophage frequencies also increased – both infiltrating (CD68+CD163+CD206-) and resident (CD68+CD163+CD206+) tissue macrophages – and these macrophages were found more near central veins over time. In contrast, CD68+CD163-CD206− trafficking myeloid cells/monocytes were detected predominantly in the periportal zone of the lobule and associated with elevated MX1 expression in that zone. Our findings identify pathologic changes within the livers of SIV infected macaques as early as 2 weeks post-infection, IFN-1 responses that are initially observed in liver macrophages, and an accumulation of macrophages associated with signs of inflammation by week 32. These studies offer quantitative and spatial insights to inform macrophage-targeted therapeutic approaches that may improve liver function during HIV infection.

Supported by grants from NIH (R01AI134630)
Using single cell spatial and transcriptional analysis to understand early host response to RNA virus infections
Authors: Arakkal Leanne , Sturdevant Gail , Robertson Shelly , Meade-White Kimberley , Germain Ronald N , Best Sonja M , Speranza Emily
Published: January 2023

The RIG-I like receptor pathway, which signals through the mitochondrial anti-viral signaling (MAVS) protein, initiates the type I interferon (IFN) response to RNA viruses such as vesicular stomatitis virus (VSV) and Ebola Virus (EBOV). To investigate the mechanism of MAVS-mediated control of VSV and EBOV infections in vivo, we utilized single cell RNA sequencing and multiplex immunohistochemistry (mIHC) in C57Bl/6J and MAVS−/− animals to understand the cellular response spatially and transcriptomically in the draining lymph nodes (dLN). Identification of virally infected cells through scRNA-Seq allowed contrast of infected cells and bystander cells. Viral quantification of EBOV and VSV samples showed an increased viral load in MAVS−/− conditions and distinct patterns of dissemination through the LN. The relationship between viral load and IFN signaling revealed a negative correlation in EBOV infected cells with no relationship in VSV infected cells highlighting the importance of viral inhibition of the IFN response in EBOV infections. To assess these patterns spatially, mIHC antibody panels have been implemented on control and experimental tissue. An in-house algorithm for cell segmentation called RAPID was applied to a controlled experiment and revealed extensive batch effects from the staining process. Current work to perform batch normalization is being carried out to better assess changes in protein marker expression. Combining information gained from the sequencing analysis with the observed spatial organization through mIHC, we aim to develop a model illustrating the role of the MAVS-dependent pathway in controlling early virus replication.

Supported by Intramural research program of the NIH
Corticosteroid Treatment Results in Enhanced susceptibility to Influenza-Associated Aspergillosis
Authors: Worthley Devyn , Lupfer Christopher R
Published: January 2023

Coinfections that occur during an influenza A virus (IAV) infection are often severe and life threatening. Since the 1918 influenza pandemic, researchers and clinicians have recognized that infection with IAV predisposes individuals to a more severe form of pneumonia resulting from secondary bacterial infections. However, coinfection following IAV infection can occur with other pathogens including other viruses and fungal pathogens. In fact, over the last two decades, numerous case reports demonstrate that influenza coinfections with Aspergillus fumigatus deserves more scrutiny as a potential source of severe respiratory disease. Here, we report that mice with Influenza-Associated Aspergillosis (IAA) develop a mild form of disease unless concomitantly treated with corticosteroids. Importantly, corticosteroid usage impairs immune function resulting in enhanced fungal growth associated with altered commensal growth in the respiratory tract. Our findings agree with case reports that suggest corticosteroids may be involved in the development of IAA.

Supported by grants from Missouri State University Graduate College.
IL-15 synergizes with IL-12 to enhance NK cell function in vivo during chronic SIV infection
Authors: Babu Hemalatha , Govindaraj Sakthivel , Wagoner Isabelle , Ali Syed A , Ibebgu Chris , Amara Rama Rao , Villinger Francois , Velu Vijayakumar
Published: January 2023

Natural killer (NK) cells play a critical role in viral immunity. During early HIV/SIV infection, they are known to protect/limit viremia at the very early stage of infection. However, during chronic HIV infection NK cells are less functional, thus elicitation of highly functional NK cells are important for HIV cure strategy. It has been shown that cytokines IL-15 and IL-12 individually can activate NK cells during SIV infection. However, the synergistic effect of these cytokines to induce NK cell function has not been studied.

We performed in vivo experiment to evaluate the effect of cytokines IL-15 (20ug/Kg)/IL-15R(100ug/kg) and IL-12 (10ug/kg) and its combination in inducing functional NK cells during chronic infection (6 weeks PI) and with no cytokine treatment as a control. Lymphocytes from blood and lymph nodes were analyzed for NK cell functions using multicolor flow cytometry.

Our in vivo data clearly indicates that IL-15/IL-15R synergistically act with IL-12 to induce proliferation (Ki-67) and cytotoxicity (Granzyme-B+) of NK cells both in blood and lymph node. Interestingly, the cytotoxic NK cells CD16+ (FCgRIII) expanded significantly in the combination group. In addition, the degranulation capacity of NK cells (IFN-gamma+ CD107a+) significantly enhanced in the combination group when compared to either cytokine group alone, suggesting that combined cytokine treatment enhanced NK cell cytotoxicity during chronic SIV infection.

Our data clearly showed that the combination of IL-15 plus IL-12 cytokine treatment increased NK cell function in vivo with proliferative and cytotoxic capacity, which is important for HIV cure strategy considering directing functional NK cells to immune privileged sites such as lymph nodes.

This work was supported in part by National Institutes of Health Grants CFAR R03, R01 HD095741-01, 1R01AI148377-01A1 (to VV), U19 AI109633 (to R.R.A.), Emory University CFAR grant P30 AI050409, NIH Office of Research Infrastructure Programs (ORIP) grants P51 OD011132 and U42 OD011023 (to YNPRC) and R24 OD010947 to the Resource for NHP Immune Reagents.
Pre-existing atopy prevents mortality from respiratory viral infection via induction of neuregulin-1
Authors: Hussain Syed-Rehan A , Rohlfing Michelle , Santoro Jennifer , Resiliac Jenny , Grayson Mitchell H
Published: January 2023

Respiratory viral infections are associated with significant morbidity and mortality. RNA viruses, such as Respiratory Syncytial Virus (RSV) have been implicated in the development of asthma. We have a high-fidelity model of respiratory viral infection. Our mouse model utilizes a natural rodent pathogen, Sendai virus (SeV) similar to RSV, which faithfully replicates in mouse airway epithelial cells. We have shown that making these mice atopic (i.e, sensitized and challenged with house dust mite extracts) before viral infection prevents development of disease and more importantly prevents mice from dying of a normally lethal dose of SeV. Further, lungs and bronchoalveolar lavage fluid of atopic mice have significant increased levels of Neuregulin-1 (NRG1), potentially due to high expression of Nrg1 in dendritic cells as determined by RNAseq of CD11c+ cells. To determine a temporal relationship between NRG1 expression and respiratory viral infection, we utilized an in vitro system of well-differentiated human bronchial epithelial cells and mouse tracheal epithelial cells. Cells were treated on the basolateral side with NRG1 (10 to 100ng) for six-days before 4000 pfu of RSV-GFP or SeV-GFP inoculation, respectively. There was marked reduction in viral spreading (quantified by reduced GFP expression) with NRG1 treatment. Interleukin-6 (IL6) expression was reduced in NRG1 treated cells at 48h PI, as well. IL6 has been implicated with cellular stress and is involved in disease pathology. Therefore, reducing IL6 might affect the clinical presentation of serious infections. In conclusion our studies show that atopy induced NRG1 plays a novel role with a therapeutic potential in severe respiratory viral infections.

Supported by Abigail Wexner Research Institute at Nationwide Children’s Hospital, Start-up fund to MHG
Nanoparticles divert monocytes away from the lungs to improve outcomes after influenza virus infection in aged mice
Authors: Elhofy Adam , Kelley William J , Chen Judy , Murthy Tushar , Xu Qichen , Boyne Michael , Goldstein Daniel
Published: January 2023

Older people exhibit dysregulated innate immunity to respiratory viral infections, including influenza and SARS-CoV-2, to increase morbidity and mortality. Nanoparticles are a potential practical therapeutic that could divert exaggerated innate immune responses away from the lungs during viral infection. However, such therapeutics have not been examined for effectiveness during respiratory viral infection, particular in aged hosts. Here, we employed a highly lethal model of influenza viral infection in vulnerable aged mice to examine the ability of biodegradable, cargo-free nanoparticles, designated ONP-302, to resolve innate immune dysfunction and improve outcomes during infection. We administered ONP-302 via intravenous injection to aged mice at day 3 post-infection when the hyperinflammatory innate immune response is already established. We found that ONP-302 treatment diverted tissue-damaging inflammatory monocytes from the lungs to the spleen without impacting viral clearance or reducing chemo-attractive signals in the infected lung. Importantly, cargo-free nanoparticles reduced lung damage and histological lung inflammation and improved gas exchange and, ultimately, the clinical outcomes in influenza-infected aged mice. ONP-302 improves outcomes in influenza-infected, vulnerable, aged mice. Thus, our study provides vital fundamental information concerning a practical therapeutic which, if translated clinically, could improve disease outcomes for vulnerable older patients suffering from respiratory viral infections.

Supported by COUR Pharmaceuticals
Controlling herpetic inflammatory lesions by inhibiting glucose metabolism- on the different outcome of using 2DG and metformin
Authors: Berber Engin , Rouse Barry T
Published: January 2023

Ocular infection with herpes simplex virus-1 (HSV-1) results in a blinding immunoinflammatory lesion. Control can be achieved by inhibiting the metabolism of cells principally involved in lesion development. Inhibiting glucose metabolism with 2DG achieves this effect but if therapy is used when replicating virus is still present, herpes simplex encephalitis (HSE) is likely to ensue. We report that using metformin to inhibit glucose metabolism can also minimize herpetic ocular lesions and this occurs without the usual complication of HSE. We try to provide a mechanistic explanation for the different outcomes. We show that the innate cell response in corneal lesions was similarly diminished by both metabolic therapies, but the effects on the composition of cellular T responses differed. Thus 2DG had more inhibitory effects than metformin on CD4 Th1 T cells, the main orchestrators of corneal lesions as well as on Foxp3+ Treg, cells responsible for controlling lesions. Metformin therapy had minimal inhibitory effects on Treg such that the representation of Treg markedly increased in comparison to Th1 cells. The responses occurring in the trigeminal ganglia, the site to which HSV spreads and then sets up permanent latency, was also investigated. The nature of the inflammatory responses at the site differed in untreated and animals treated with 2DG or metformin. Thus, both therapies resulted in diminished inflammatory responses particularly the innate cell composition. The inhibitory effects of 2DG against the CD8 content of the inflammatory reaction (cells advocated as involved in containing latency and stopping virus from disseminating to the brain) was significantly greater than was the metformin therapy.

Supported by grants from NIH (R21AI142862 and R01EY005093)
Role of exosomal BTLA/HVEM signaling in HTLV-1 associated diseases and associated T-cell dysfunction.
Authors: Joseph Julie , Rao Abhishek , Connors Jennifer , Stoffel Victoria , Nootenboom Julianne , Rahmani Benjamin , Haddad Elias , Carey Alison , Jain Pooja
Published: January 2023

Immune checkpoint (ICP) mediators play pivotal roles in regulating a broad spectrum of immune responses against cancer and infectious diseases. In previous studies we have established the role of negative checkpoint receptors (NCRs, PD-1, TIGIT, LAG-3, etc.) in determining the quality of anti-HTLV-1 cytolytic (CTL) response in controlling proviral load and maintaining a asymptomatic state in majority of infected individuals. Interestingly, ICPs can be released in the soluble form and carried on the surface of small extracellular vesicles (50–200 nm) known as exosomes that possess immunomodulatory activity. Consequently, we profiled ICPs in isolated exosomes and culture media of HTLV-1 cell lines representing both HAM/TSP and ATLL. High levels of BTLA (B-and T-Lymphocyte Attenuator) and its ligand HVEM (Herpes virus entry mediator) along with PD-1 (Programmed cell death receptor-1) and its ligand PD-L2 (not PD-L1) were observed in soluble and exosomal forms as well in the sera of HAM patients that carry high proviral load and viral proteins such as Tax. HTLV-1 infection, Tax protein and/or IFNy could serve as causative mechanism for increased ICP levels in HAM patients. Indeed, treatment with anti-retroviral drugs significantly reduced ICP levels confirming specificity of our observations. Functionality of exosomal BTLA/HVEM is being validated in the ongoing studies along with their direct role in regulating CD8 T-cell functions and cytolytic potential upon blockade to provide a novel therapeutic target for HAM/TSP and other neuroinflammatory diseases.

Supported by grants from NIH: 1R01NS0971-47, T32-MH079785
Radioresistant cells in STING gain-of-function mice initiate lymphocyte dependent lung inflammation and IFNγ dependent mortality
Authors: Gao Kevin MingJie , Marshak-Rothstein Ann , Fitzgerald Katherine A
Published: January 2023

Pediatric patients with gain-of-function (GOF) mutations in the cytosolic dsDNA sensing adaptor STING develop an autoinflammatory syndrome known as STING Associated Vasculopathy with onset in Infancy (SAVI). SAVI patients show elevated interferon signature genes and suffer from interstitial lung disease (ILD) with lymphocyte predominate bronchus associated lymphoid tissue (BALT). We genetically engineered SAVI mice (STING V154M or VM) that recapitulates human disease, including lymphocyte rich ILD, and found by genetic ablation that lymphocytes are required for lung pathology. However, while ablation of either T or B lymphocytes prolongs survival, lung immune aggregates persist, indicating either can independently be recruited as BALT. Using radiation chimeras, we found that hematopoietic stem cell (HSC) derived STING GOF was neither sufficient nor required for VM lung disease, suggesting a role for lung radioresistant parenchymal and stromal cell STING GOF in SAVI. We identified lung endothelia as putative radioresistant cells which express STING and confirmed expression by IF microscopy and FACS. FACS analysis also revealed SAVI lung endothelia upregulate MHCII, but only when sufficient for IFNgR. VM T cells produce IFNg, and IFNgR deficiency was sufficient to prevent endothelial MHCII upregulation and attenuate mortality. However, HSC STING GOF was not required for endothelial MHCII upregulation, suggesting stromal intrinsic STING GOF initiates this outcome. These findings suggest that activation of STING in stromal and parenchymal cells play a key role the initiation of lung inflammation in patients with connective tissue disease ILD.

Supported by: T32 GM107000 (MSTP); T32 AI132152 (KMG); F30 HL154674 (KMG); AI128358 (AMR/KAF); Lupus Research Alliance
The role of NKG2D signaling in NOD diabetes is affected by the microbiota
Authors: Markiewicz Mary A , Bedrosian Zoe K , Ruark Elizabeth , Krausz Kelsey
Published: January 2023

The drivers of type 1 diabetes are islet-specific T cells. Understanding the mechanisms by which these cells are activated is critical to developing better strategies to inhibit the disease. Signaling through NKG2D is implicated in this process, but its role is unclear owing to conflicting results from studies performed to date. Using non-obese diabetic (NOD) mice genetically deficient in NKG2D, we demonstrate that the effect of NKG2D signaling on NOD diabetes varies with housing conditions, with NKG2D signaling having the greatest role in germ-free mice. We show this signaling does not occur through interaction with ligands expressed within pancreatic islets, as we demonstrate that NKG2D signaling within the islets instead inhibits NOD diabetes development. Specifically, we show that NOD mice we generated with a genetic deficiency in the endogenous NKG2D ligand expressed in the islets, H60a, have increased diabetes development, whereas H60a-deficient NOD mice expressing the NKG2D ligand RAE1ɛ in β-islet cells (via a transgene) have reduced diabetes. Further, unlike the decrease in diabetes observed in germ-free NKG2D-deficient mice, there was no difference in diabetes development between germ-free H60a-deficient and wild-type NOD mice. Taken together, these data demonstrate that (1) the composition of the microbiota affects how significant a role NKG2D signaling plays in promoting NOD diabetes and (2) this signaling occurs outside of the pancreatic islets. We further demonstrate that antigen presenting cells (APCs) in the pancreatic lymph nodes of both specific pathogen free (SPF) and germ-free NOD mice express members of the NKG2D ligand family RAE1, indicating this may be the site of the diabetes-promoting NKG2D signaling.

Supported by grants from DOD (PR181543), Emilie Rosebud Diabetes Research Foundation, KUMC Diabetes Institute, and Beatson Foundation (2020-004). In addition, the University of Kansas Medical Center Flow Core was utilized, which was funded in part by NIH (P30 GM122731; P30 GM103326; P30 CA168524).