Novel prime-boost immunization strategies are required to control the global Tuberculosis (TB) pandemic, which claims approximately 3 lives every minute. Here, we have generated an immunogenic complex against Mycobacterium tuberculosis (M.tb), consisting of promiscuous T cell epitopes (M.tb peptides) and TLR ligands assembled in liposomes. Interestingly, this complex (PTLs; peptide-TLR agonist-liposomes) induced significant activation of CD4+ T cells and IFNγ production in the PBMCs derived from PPD+ healthy individuals as compared to PPD- controls. Furthermore, intranasal delivery of PTLs significantly reduced the bacterial burden in the infected mice by inducing M.tb specific polyfunctional (IFNγ+IL17+TNFα+IL2+) immune responses and long-lasting central memory responses thereby reducing the risk of TB recurrence in DOTS treated infected animals. The transcriptome analysis of peptide-stimulated immune cells unveiled the molecular basis of enhanced protection. Furthermore, PTLs immunization significantly boosted the BCG-primed immune responses against TB. The greatly enhanced efficacy of BCG-PTLs vaccine model in controlling pulmonary TB projects PTLs as an adjunct vaccine against TB.
Santosh Kumar, Ashima Bhaskar, Gautam Patnaik, Chetan Sharma, Dhiraj K. Singh, Sandeep Kaushik, Shivam Chaturvedi, Gobardhan Das, Ved Prakash Dwivedi
Hepatitis B virus (HBV)-specific CD8+ T cells fail to acquire effector functions after priming in the liver, but the molecular basis for the dysfunctionality is poorly understood. By comparing the gene expression profile of intrahepatically primed, dysfunctional HBV-specific CD8+ T cells with that of systemically primed, functional effector counterparts, we found that the expression of interferon-stimulated genes (ISGs) is selectively suppressed in the dysfunctional CD8+ T cells. The ISG suppression was associated with impaired phosphorylation of STAT1 in response to IFNα treatment. Importantly, a strong induction of type interferons (IFN-Is) in the liver facilitated the functional differentiation of intrahepatically primed HBV-specific CD8+ T cells in association with the restoration of ISGs expression in the T cells. These results suggest that intrahepatic priming suppresses IFN-I signaling in CD8+ T cells, which may contribute to the dysfunctionality. The data also suggest a therapeutic value of the robust induction of intrahepatic IFN-Is for the treatment of chronic HBV infection.
Keigo Kawashima, Masanori Isogawa, Masaya Onishi, Ian Baudi, Satoru Saito, Atsushi Nakajima, Takashi Fujita, Yasuhito Tanaka
CIS43 is a potent neutralizing human monoclonal antibody (mAb) that targets a highly conserved ‘junctional’ epitope in the Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP). Enhancing the durability of CIS43 in vivo will be important for clinical translation. Here, two approaches were used to improve the durability of CIS43 in vivo while maintaining potent neutralization. First, the Fc domain was modified with the “LS” mutations (CIS43LS) to increase CIS43 binding affinity for the neonatal Fc receptor (FcRn). CIS43LS and CIS43 showed comparable in vivo protective efficacy. CIS43LS had nine to thirteen-fold increased binding affinity for human (6.2 nM vs. 54.2 nM) and rhesus (25.1 nM vs. 325.8 nM) FcRn at endosomal pH 6.0 compared to CIS43. Importantly, the half-life of CIS43LS in macaques increased from 22 days to 39 days compared to CIS43. The second approach for sustaining antibody levels of CIS43 in vivo is through adeno-associated virus (AAV) expression. Mice administered once with AAV expressing CIS43 had sustained antibody levels of ~ 300 μg/mL and mediated protection against sequential malaria challenges up to 36 weeks. Based on these data, CIS43LS has advanced to Phase I clinical trials and AAV delivery provides a potential next generation approach.
Neville Kielau Kisalu, Lais Da Silva Pereira, Keenan J. Ernste, Yevel Flores-Garcia, Azza H. Idris, Mangaiarkarasi Asokan, Marlon Dillon, Scott MacDonald, Wei Shi, Xuejun Chen, Amarendra Pegu, Arne Schön, Fidel Zavala, Alejandro B. Balazs, Joseph R. Francica, Robert A. Seder
Background. Clostridioides difficile is a major cause of healthcare-associated diarrhea. Severity ranges from mild to life-threatening, but this variability remains poorly understood. Microbiological diagnosis of C. difficile infection (CDI) is straightforward, but offers little insight into the patient's prognosis, nor into pathophysiological determinants of clinical trajectory. The aim of this study was to discover host-derived, CDI-specific, fecal biomarkers involved in disease severity. Methods. Subjects without and with diarrhea were recruited. CDI was established by commercial, diagnostic real-time PCR assay of tcdB. CDI severity was based on IDSA/SHEA criteria. We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) approach to identify host-derived protein biomarkers from stool and applied it to diagnostic samples for cohort-wise comparison (CDI-negative vs. non-severe CDI vs. severe CDI). Selected biomarkers were orthogonally confirmed and subsequently verified in a CDI mouse model. Results. We identified a protein signature from stool, consisting of alpha-2-macroglobulin (A2M), matrix metalloproteinase-7 (MMP7) and alpha-1-antitrypsin (A1AT), that not only discriminates CDI-positive samples from non-CDI ones, but is potentially associated with disease severity. In the mouse model, this signature with the murine homologs of the corresponding proteins was also identified. Conclusions. A2M, MMP7 and A1AT serve as biomarkers in patients with CDI and define novel components of the host response that may determine disease severity.
Makan Golizeh, Kaitlin Winter, Lucie Roussel, Marija Landekic, Melanie Langelier, Vivian G. Loo, Momar Ndao, Donald C. Vinh
Peptidylarginine deiminases (PADs) are a family of calcium-dependent enzymes that are involved in a variety of human disorders, including cancer and autoimmune diseases. Although targeting PAD4 has shown no benefit in sepsis, the role of PAD2 remains unknown. Here, we report that PAD2 is engaged in sepsis and sepsis-induced acute lung injury in both human patients and mice. Pad2–/– or selective inhibition of PAD2 by a small molecule inhibitor increased survival and improved overall outcomes in mouse models of sepsis. Pad2 deficiency decreased neutrophil extracellular trap (NET) formation. Importantly, Pad2 deficiency inhibited Caspase-11–dependent pyroptosis in vivo and in vitro. Suppression of PAD2 expression reduced inflammation and increased macrophage bactericidal activity. In contrast to Pad2–/–, Pad4 deficiency enhanced activation of Caspase-11–dependent pyroptosis in BM-derived macrophages and displayed no survival improvement in a mouse sepsis model. Collectively, our findings highlight the potential of PAD2 as an indicative marker and therapeutic target for sepsis.
Yuzi Tian, Shibin Qu, Hasan B. Alam, Aaron M. Williams, Zhenyu Wu, Qiufang Deng, Baihong Pan, Jing Zhou, Baoling Liu, Xiuzhen Duan, Jianjie Ma, Santanu Mondal, Paul R. Thompson, Kathleen A. Stringer, Theodore J. Standiford, Yongqing Li
There is no cure for the more than 270 million people chronically infected with HBV. Nucleos(t)ide analogs (NUCs), the mainstay of anti-HBV treatment, block HBV reverse transcription. NUCs do not eliminate the intranuclear covalently closed circular DNA (cccDNA), from which viral RNAs, including pregenomic RNA (pgRNA), are transcribed. A key gap in designing a cure is understanding how NUCs affect HBV replication and transcription because serum markers yield an incomplete view of intrahepatic HBV. We applied single-cell laser capture microdissection and droplet digital PCR to paired liver biopsies collected from 5 HBV/HIV-coinfected persons who took NUCs over 2–4 years. From biopsy 1 to 2, proportions of HBV-infected hepatocytes declined with adherence to NUC treatment (P < 0.05); we extrapolated that eradication of HBV will take over 10 decades with NUCs in these participants. In individual hepatocytes, pgRNA levels diminished 28- to 73-fold during NUC treatment, corresponding with decreased tissue HBV core antigen staining (P < 0.01). In 4 out of 5 participants, hepatocytes with cccDNA but undetectable pgRNA (transcriptionally inactive) were present, and these were enriched in 3 participants during NUC treatment. Further work to unravel mechanisms of cccDNA transcriptional inactivation may lead to therapies that can achieve this in all hepatocytes, resulting in a functional cure.
Ashwin Balagopal, Tanner Grudda, Ruy M. Ribeiro, Yasmeen S. Saad, Hyon S. Hwang, Jeffrey Quinn, Michael Murphy, Kathleen Ward, Richard K. Sterling, Yang Zhang, Alan S. Perelson, Mark S. Sulkowski, William O. Osburn, Chloe L. Thio
Staphylococcus aureus is prevalent in surgical site infections (SSI) and leads to death in approximately 1% of patients. Phase IIB/III clinical trial results have demonstrated that vaccination against the iron-regulated surface determinant protein B (IsdB) is associated with an increased mortality rate in patients with SSI. Thus, we hypothesized that S. aureus induces nonneutralizing anti-IsdB antibodies, which facilitate bacterial entry into leukocytes to generate “Trojan horse” leukocytes that disseminate the pathogen. Since hemoglobin (Hb) is the primary target of IsdB, and abundant Hb-haptoglobin (Hb-Hp) complexes in bleeding surgical wounds are normally cleared via CD163-mediated endocytosis by macrophages, we investigated this mechanism in vitro and in vivo. Our results demonstrate that active and passive IsdB immunization of mice renders them susceptible to sepsis following SSI. We also found that a multimolecular complex containing S. aureus protein A–anti-IsdB–IsdB–Hb-Hp mediates CD163-dependent bacterial internalization of macrophages in vitro. Moreover, IsdB-immunized CD163–/– mice are resistant to sepsis following S. aureus SSI, as are normal healthy mice given anti-CD163–neutralizing antibodies. These genetic and biologic CD163 deficiencies did not exacerbate local infection. Thus, anti-IsdB antibodies are a risk factor for S. aureus sepsis following SSI, and disruption of the multimolecular complex and/or CD163 blockade may intervene.
Kohei Nishitani, Masahiro Ishikawa, Yugo Morita, Noriaki Yokogawa, Chao Xie, Karen L. de Mesy Bentley, Hiromu Ito, Stephen L. Kates, John L. Daiss, Edward M. Schwarz
Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin converting-enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of two viral based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus, and a spike pseudotyped viral-vector-based assay.
Kento T. Abe, Zhijie Li, Reuben Samson, Payman Samavarchi-Tehrani, Emelissa J. Valcourt, Heidi Wood, Patrick Budylowski, Alan P. Dupuis II, Roxie C. Girardin, Bhavisha Rathod, Jenny Wang, Miriam Barrios-Rodiles, Karen Colwill, Allison McGeer, Samira Mubareka, Jennifer L. Gommerman, Yves Durocher, Mario Ostrowski, Kathleen A. McDonough, Michael A. Drebot, Steven J. Drews, James M. Rini, Anne-Claude Gingras
The emergence of SARS-CoV-2 has created an international health crisis. Small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection due to low affinity binding to the murine angiotensin-converting enzyme 2 (ACE2) protein. Here we evaluated the pathogenesis of SARS-CoV-2 in male and female mice expressing the human ACE2 gene under the control of the keratin 18 promotor. In contrast to non-transgenic mice, intranasal exposure of K18-hACE2 animals to two different doses of SARS-CoV-2 resulted in acute disease including weight loss, lung injury, brain infection and lethality. Vasculitis was the most prominent finding in the lungs of infected mice. Transcriptomic analysis from lungs of infected animals revealed increases in transcripts involved in lung injury and inflammatory cytokines. In the lower dose challenge groups, there was a survival advantage in the female mice with 60% surviving infection whereas all male mice succumbed to disease. Male mice that succumbed to disease had higher levels of inflammatory transcripts compared to female mice. This is the first highly lethal murine infection model for SARS-CoV-2. The K18-hACE2 murine model will be valuable for the study of SARS-CoV-2 pathogenesis and the assessment of MCMs.
Joseph W. Golden, Curtis R. Cline, Xiankun Zeng, Aura R. Garrison, Brian D. Carey, Eric M. Mucker, Lauren E. White, Joshua D. Shamblin, Rebecca L. Brocato, Jun Liu, April M. Babka, Hypaitia B. Rauch, Jeffrey M. Smith, Bradley S. Hollidge, Collin Fitzpatrick, Catherine V. Badger, Jay W. Hooper
Background: Control of the tuberculosis (TB) pandemic remains hindered, in part, by a lack of simple and accurate measures of treatment efficacy. Current gold standard markers rely on sputum-based assays that are slow and challenging to implement. Previous work identified urinary N1, N12-diacetylspermine (DiAcSpm), neopterin, hydroxykynurenine, N-acetylhexosamine, ureidopropionic acid, sialic acid, and m/z 241.0903 as potential biomarkers of active pulmonary TB (ATB). Here, we evaluated their ability to serve as biomarkers of TB treatment response and mycobacterial load. Methods: We analyzed urine samples prospectively collected from two cohorts with ATB: 34 participants from African countries treated with first line TB therapy (HRZE) and followed for one year, and 35 participants from Haiti treated with either HRZE or an experimental drug followed for 14 days. Blinded samples were analyzed by untargeted high-performance liquid chromatography-coupled-time of flight-mass spectrometry. Results: Urinary levels of all seven molecules exhibited significant decreases by week 26 of successful treatment (p=0.01-p<0.0001), and positive correlations with sputum mycobacterial load (p<0.0001). Urinary levels of DiAcSpm exhibited significant decreases in participants treated with HRZE as early as 14 days (p<0.0001) but were unchanged in participants receiving ineffective therapy (p=0.14). Conclusion: Reductions in urinary DiAcSpm, neopterin, hydroxykynurenine, N-acetylhexosamine, ureidopropionic acid, sialic acid, and m/z 241.0903 correlated with successful anti-TB treatment and sputum mycobacterial load. Levels of DiAcSpm exhibited reductions capable of differentiating treatment success from failure as early as two weeks after the initiation of chemotherapy, commending its further development as a potentially simple, non-invasive biomarker of treatment response and bacterial load. Funding: This work was supported by the Clinical and Translational Science Center at Weill Cornell College of Medicine (NIH/NCATS 1 UL1 TR002384-02 and KL2TR000458), the Department of Defense (PR170782), the National Institute of Allergy and Infectious Disease grants (NIAID T32AI007613-16, K24 AI098627 and K23 AI131913), the NIH Fogarty International Center grants (R24 TW007988 and TW010062), the National Institute of Health grant (R01 GM135926 ), the Abby and Howard P. Milstein Program in Chemical Biology and Translational Medicine, and the Tuberculosis Research Units Networks (TBRU-N, AI111143).
Qianjing Xia, Myung Hee Lee, Kathleen F. Walsh, Kathrine McAulay, James M. Bean, Daniel W. Fitzgerald, Kathryn M. Dupnik, Warren D. Johnson, Jean W. Pape, Kyu Y. Rhee, Flonza Isa
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