The saponin monophosphoryl lipid A nanoparticle adjuvant induces dose-dependent HIV vaccine responses in non-human primates

• Text
• PDF

Abstract

The induction of durable protective immune responses is the main goal of prophylactic vaccines, and adjuvants play a role as drivers of such responses. Despite advances in vaccine strategies, a safe and effective HIV vaccine remains a significant challenge. The use of an appropriate adjuvant is crucial to the success of HIV vaccines. Here we assessed the saponin/MPLA nanoparticle (SMNP) adjuvant with an HIV envelope (Env) trimer, evaluating the safety and impact of multiple variables including adjuvant dose (16-fold dose range), immunization route, and adjuvant composition on the establishment of Env-specific memory T and B cell responses (TMem and BMem) and long-lived plasma cells in non-human primates (NHPs). Robust BMem were detected in all groups, but a 6-fold increase was observed in the highest SMNP dose group vs. the lowest dose group. Similarly, stronger vaccine responses were induced in the highest SMNP dose for CD40L+OX40+ CD4 TMem (11-fold), IFN-γ+ CD4 TMem (15-fold), IL21+ CD4 TMem (9-fold), circulating TFH (3.6-fold), bone marrow plasma cells (7-fold), and binding IgG (1.3-fold). Substantial tier-2 neutralizing antibodies were only observed in the higher SMNP dose groups. These investigations highlight the dose-dependent potency of SMNP in NHPs, which are relevant for human use and next-generation vaccines.

Authors

Parham Ramezani-Rad, Ester Marina-Zárate, Laura Maiorino, Amber Myers, Katarzyna Kaczmarek Michaels, Ivan S. Pires, Nathaniel I. Bloom, Mariane B. Melo, Ashley A. Lemnios, Paul G. Lopez, Christopher A. Cottrell, Iszac Burton, Bettina Groschel, Arpan Pradhan, Gabriela Stiegler, Magdolna Budai, Daniel Kumar, Sam Pallerla, Eddy Sayeed, Sangeetha L. Sagar, Sudhir Pai Kasturi, Koen K.A. Van Rompay, Lars Hangartner, Andreas Wagner, Dennis R. Burton, William R. Schief, Shane Crotty, Darrell J. Irvine

Cathepsin K cleavage of Angiopoietin-2 creates detrimental Tie2 antagonist fragments in sepsis

• Text
• PDF

Abstract

Elevated Angiopoietin-2 is associated with diverse inflammatory conditions including sepsis, a leading global cause of mortality. During inflammation, Angiopoietin-2 antagonizes the endothelium-enriched receptor Tie2 to destabilize the vasculature. In other contexts, Angiopoietin-2 stimulates Tie2. The basis for context-dependent antagonism remains incompletely understood. Here we show that inflammation-induced proteolytic cleavage of Angiopoietin-2 converts this ligand from Tie2 agonist to antagonist. Conditioned media from stimulated macrophages induced endothelial Angiopoietin-2 secretion. Unexpectedly, this was associated with reduction of the 75 kDa full-length protein and appearance of new 25 and 50 kDa C-terminal fragments. Peptide sequencing proposed cathepsin K as a candidate protease. Cathepsin K was necessary and sufficient to cleave Angiopoietin-2. Recombinant 25 and 50 kDa Angiopoietin-2 fragments (cANGPT225, cANGPT250) bound and antagonized Tie2. Cathepsin K inhibition with the Phase-3 small molecule inhibitor odanacatib improved survival in distinct murine sepsis models. Full-length Angiopoietin-2 enhanced survival in endotoxemic mice administered odanacatib and, conversely, increased mortality in the drug’s absence. Odanacatib’s benefit was reversed by heterologous cANGPT225. Septic humans accumulated circulating Angiopoietin-2 fragments, which were associated with adverse outcomes. These results identify cathepsin K as a candidate marker of sepsis and a proteolytic mechanism for the conversion of Angiopoietin-2 from Tie2 agonist to antagonist with therapeutic implications for inflammatory conditions associated with Angiopoietin-2 induction.

Authors

Takashi Suzuki, Erik Loyde, Sara Chen, Valerie Etzrodt, Temitayo O. Idowu, Amanda J. Clark, Marie Christelle Saade, Brenda Mendoza Flores, Shulin Lu, Gabriel Birrane, Vamsidhara Vemireddy, Benjamin Seeliger, Sascha David, Samir M. Parikh

CRISPR-mediated detection of Pneumocystis transcripts in bronchoalveolar, oropharyngeal, and serum specimens for Pneumocystis pneumonia diagnosis

• Text
• PDF

Abstract

BACKGROUND.Pneumocystis jirovecii pneumonia (PCP) is a leading cause of fungal pneumonia, but its diagnosis primarily relies on invasive bronchoalveolar lavage (BAL) specimens that are difficult to obtain. Oropharyngeal swabs and serum could improve the PCP diagnostic workflow, and we hypothesized that CRISPR could enhance assay sensitivity to allow robust P. jirovecii diagnosis using swabs and serum. Herein we describe the development of an ultrasensitive RT-PCR-coupled CRISPR assay with high active-infection specificity in infant swabs and adult BAL and serum. METHODS. Mouse analyses employed an RT-PCR CRISPR assay to analyze P. murina transcripts in wild-type and Rag2–/– mouse lung RNA, BAL, and serum at 2-, 4-, and 6-weeks post-infection. Human studies used an optimized RT-PCR CRISPR assay to detect P. jirovecii transcripts in infant oropharyngeal swab samples, adult serum, and adult BAL specimens from P. jirovecii-infected and P. jirovecii-non-infected patients. RESULTS. The P. murina assays sensitively detected Pneumocystis RNA in the serum of infected mice throughout infection. Oropharyngeal swab CRISPR assay results identified infants infected with P. jirovecii with greater sensitivity (96.3% vs. 66.7%) and specificity (100% vs. 90.6%) than RT-qPCR compared to mtLSU standard marker, and CRISPR results achieved higher sensitivity than RT-qPCR results (93.3% vs. 26.7%) in adult serum specimens. CONCLUSION. Since swabs are routinely collected in pediatric pneumonia patients and serum is easier to obtain than BAL, this assay approach could improve the accuracy and timing of pediatric and adult Pneumocystis diagnosis by achieving specificity for active infection and potentially avoiding the requirement for BAL specimens.

Authors

Brady M. Youngquist, Ayanda Trevor Mnguni, Dora Pungan, Rachel P.J. Lai, Guixiang Dai, Chun Fai Ng, Amy Samson, Yasmean Abdelgaliel, Christopher J. Lyon, Bo Ning, Shahid Husain, Sean Wasserman, Jay K. Kolls, Tony Y. Hu

Sequential carbonyl derivatives and hydrazone adduct formation on myeloperoxidase contribute to development of ANCA-vasculitis

• Text
• PDF

Abstract

Drug-induced autoimmune diseases are increasingly recognized although mechanistic insight into disease causation is lacking. Hydralazine exposure has been linked to autoimmune diseases, including anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis. Our hypothesis posits that hydralazine covalently binds to myeloperoxidase (MPO), triggering the autoimmune response in ANCA vasculitis. We in vitro observed formation of carbonyl derivatives on amine groups in the presence of acrolein. This facilitated the subsequent binding of hydralazine to heme-containing proteins, including MPO, via a Michael addition. Our studies demonstrated that carbonyl derivatives and hydrazone adducts induce conformational changes in the MPO heavy chain, potentially changing its immunogenicity. We identified hydrazone adducts on circulating MPO in patients with hydralazine-associated ANCA vasculitis. These patients exhibited elevated anti-MPO IgM levels, while anti-MPO IgG levels were comparable between hydralazine-associated and non-hydralazine-associated vasculitis patients. IgM isolated from hydralazine-associated MPO ANCA patients demonstrated a heightened affinity to hydralazine-modified MPO and activated neutrophil-like HL-60 cells. Hydralazine-modified MPO was pathogenic, as demonstrated by splenocyte transfer in a mouse model of ANCA vasculitis. Our findings unveil a mechanism of drug-induced autoimmunity wherein stepwise chemical modifications of MPO lead to conformational changes and hydrazone adduct formation producing a neoantigen to which pathogenic autoantibodies are generated.

Authors

Gang Xi, Elizabeth A. Mclnnis, Olivier Lardinois, Peiqi Hu, John S. Poulton, Meghan E. Free, Dhruti P. Chen, Evan M. Zeitler, Eveline Y. Wu, Nicole M. Orzechowski, Vimal K. Derebail, J. Charles Jennette, Ronald J. Falk

Identification of potent biparatopic antibodies targeting FGFR2 fusion driven cholangiocarcinoma

• Text
• PDF

Abstract

Translocations involving FGFR2 gene fusions are common in cholangiocarcinoma and predict response to FGFR kinase inhibitors. However, response rates and durability are limited due to the emergence of resistance, typically involving FGFR2 kinase domain mutations, and to sub-optimal dosing, relating to drug adverse effects. Here, we develop biparatopic antibodies targeting the FGFR2 extracellular domain (ECD), as candidate therapeutics. Biparatopic antibodies can overcome drawbacks of bivalent monospecific antibodies, which often show poor inhibitory or even agonist activity against oncogenic receptors. We show that oncogenic transformation by FGFR2 fusions requires an intact ECD. Moreover, by systematically generating biparatopic antibodies targeting distinct epitope pairs in FGFR2 ECD, we identified antibodies that effectively block signaling and malignant growth driven by FGFR2-fusions. Importantly, these antibodies demonstrate efficacy in vivo, synergy with FGFR inhibitors, and activity against FGFR2 fusions harboring kinase domain mutations. Thus, biparatopic antibodies may serve as an innovative treatment option for patients with FGFR2-altered cholangiocarcinoma.

Authors

Saireudee Chaturantabut, Sydney Oliver, Dennie T. Frederick, Jiwan J. Kim, Foxy P. Robinson, Alessandro Sinopoli, Tian-Yu Song, Yao He, Yuan-Chen Chang, Diego J. Rodriguez, Liang Chang, Devishi Kesar, Meilani Ching, Ruvimbo Dzvurumi, Adel Atari, Yuen-Yi Tseng, Nabeel Bardeesy, William R. Sellers

Megakaryocytes transfer mitochondria to bone marrow mesenchymal stromal cells to lower platelet activation

• Text
• PDF

Abstract

Newly produced platelets acquire a low activation state but whether the megakaryocyte plays a role in this outcome has not been fully uncovered. Mesenchymal stem cells (MSCs) were previously shown to promote platelet production and lower platelet activation. We found healthy megakaryocytes transfer mitochondria to MSCs mediated by Connexin 43 (Cx43) gap junctions on MSCs, which leads to platelets at a low energetic state with increased LYN activation, characteristic of resting platelets. On the contrary, MSCs have a limited ability to transfer mitochondria to megakaryocytes. Sickle cell disease (SCD) is characterized by hemolytic anemia and results in heightened platelet activation, contributing to numerous disease complications. Platelets in SCD mice and human patient samples had a heightened energetic state with increased glycolysis. MSC exposure to heme in SCD led to decreased Cx43 expression and a reduced ability to uptake mitochondria from megakaryocytes. This prevented LYN activation in platelets and contributed to increased platelet activation at steady state. Altogether, our findings demonstrate an effect of hemolysis in the microenvironment leading to increased platelet activation in SCD. These findings have the potential to inspire new therapeutic targets to relieve thrombosis-related complications of SCD and other hemolytic conditions.

Authors

Chengjie Gao, Yitian Dai, Paul A. Spezza, Paul Boasiako, Alice Tang, Giselle Rasquinha, Hui zhong, Bojing Shao, Yunfeng Liu, Patricia A. Shi, Cheryl A. Lobo, Xiuli An, Anqi Guo, William B. Mitchell, Deepa Manwani, Karina Yazdanbakhsh, Avital Mendelson

A multicenter prospective clinical trial reveals cell free DNA methylation markers for early esophageal cancer

• Text
• PDF

Abstract

BACKGROUND. Current methods for detecting esophageal cancer (EC) are generally invasive or exhibit limited sensitivity and specificity, especially for the identification of early-stage tumors. METHODS. We identified potential methylated DNA markers (MDM) from multiple genomic regions in a discovery cohort and a diagnostic model was developed and verified in a model-verification cohort of 297 participants. The accuracy of the MDM panel was validated in a multicenter, prospective cohort (n = 1429). The clinical performance of identified MDMs were compared with current tumor-associated protein markers. RESULTS. From 31 significant differentially methylated EC-associated regions identified in the marker discovery, we trained and validated a 3-MDM diagnostic model that could discriminate among EC patients and Non-EC volunteers in a multicenter clinical prospective cohort with a sensitivity of 85.5% and a specificity of 95.3%. This panel showed higher sensitivity in diagnosing early-stage tumors, with sensitivities of 56% for Stage 0 and 77% for Stage I, comparing with the performance of current biochemical markers. In population with high risk for EC, the sensitivity and specificity are 85.68% and 93.61% respectively. CONCLUSION. The assessment of tumor-associated methylation status in blood samples can facilitate non-invasive, and reliable diagnosis of early-stage EC, which warrants further development to expand screening and reduce mortality rates. TRIAL REGISTRATION NUMBER. ChiCTR2400083525.

Authors

Ruixiang Zhang, Yongzhan Nie, Xiaobing Chen, Tao Jiang, Jinhai Wang, Yuhui Peng, Guangpeng Zhou, Yong Li, Lina Zhao, Beibei Chen, Yunfeng Ni, Yan Cheng, Yiwei Xu, Zhenyu Zhu, Xianchun Gao, Zhen Wu, Tianbao Li, Jie Zhao, Cantong Liu, Gang Zhao, Jiakuan Chen, Jing Zhao, Gang Ji, Xiaoliang Han, Jie He, Yin Li

An inherited predisposition allele promotes gastric cancer via enhancing deubiquitination-mediated activation of epithelial-to-mesenchymal transition signaling

• Text
• PDF

Abstract

Genome-wide human genetic studies have identified inherited cis-regulatory loci variants that predispose to cancers. However, the mechanisms by which these germline variants influence cancer progression, particularly through gene expression and proteostasis control, remain unclear. By analyzing genomic data from a gastric cancer (GC) case-control study (2,117 individuals), focusing on the ubiquitin-specific protease (USP) family, we identify the single nucleotide polymorphism (SNP) rs72856331 (G>A) in the promoter region of the proto-oncogene USP47 as a putative susceptibility allele for GC (OR = 0.78, P = 0.015). Mechanistically, the risk allele G is associated with enhanced USP47 expression, mediated by altered recruitment of the transcription factor GLI3 and changes in the epigenetic status at promoter. CRISPR/Cas9-mediated single-nucleotide conversion into risk allele G results in increased GLI3 binding and subsequent USP47 upregulation. The depletion of GLI3 results in a reduction of cancer-related phenotypes, similar to those observed following USP47 knockdown. Furthermore, we identify Snai1 as a deubiquitination target of USP47, explaining USP47-dependent activation of epithelial-mesenchymal transition pathway and tumor progression. Our findings identify an important genetic predisposition that implicates the perturbation of transcription and proteostasis programs in GC, offering insights into prevention and therapeutic strategies for genetically stratified patients.

Authors

Bolin Tao, Zhenning Wang, Xuanyi Wang, Aixia Song, Jiaxian Liu, Jianan Wang, Qin Zhang, Zhaolin Chen, Zixian Wang, Wenjie Xu, Menghong Sun, Yanong Wang, Ping Zhang, Tao Xu, Gong-Hong Wei, Fei Xavier Chen, Mengyun Wang

An endothelial SOX18-mevalonate pathway axis enables repurposing of statins for infantile hemangioma

• Text
• PDF

Abstract

Infantile hemangioma (IH) is the most common tumor in children and a paradigm for pathological vasculogenesis, angiogenesis, and regression. Propranolol, the mainstay treatment, inhibits IH vessel formation via a β-adrenergic receptor independent off-target effect of its R(+) enantiomer on the endothelial SRY box transcription factor 18 (SOX18). Transcriptomic profiling of patient-derived hemangioma stem cells (HemSC) uncovered the mevalonate pathway (MVP) as a target of R(+) propranolol. Loss and gain of function of SOX18 confirmed it is both necessary and sufficient for R(+) propranolol suppression of the MVP, including regulation of sterol regulatory element binding protein 2 (SREBP2) and the rate-limiting enzyme HMG-CoA reductase (HMGCR). AThe biological relevance of the endothelial SOX18-MVP axis in IH patient tissue was demonstrated by nuclear co-localization of SOX18 and SREBP2. Functional validation in a preclinical IH xenograft model revealed that statins – competitive inhibitors of HMGCR – efficiently suppress IH vessel formation. We propose an novel endothelial SOX18-MVP-axis as a central regulator of IH pathogenesis and suggest statin repurposing to treat IH. The pleiotropic effects of R(+) propranolol and statins along the SOX18-MVP axis to disable an endothelial-specific program may have therapeutic implications for other vascular disease entities involving pathological vasculogenesis and angiogenesis.

Authors

Annegret Holm, Matthew S. Graus, Jill Wylie-Sears, Jerry Wei Heng Tan, Maya Alvarez-Harmon, Luke Borgelt, Sana Nasim, Long Chung, Ashish Jain, Mingwei Sun, Liang Sun, Pascal Brouillard, Ramrada Lekwuttikarn, Yanfei Qi, Joyce Teng, Miikka Vikkula, Harry Kozakewich, John B. Mulliken, Mathias Francois, Joyce Bischoff

Asparagine drives immune evasion in bladder cancer via RIG-I stability and type I IFN signaling

• Text
• PDF

Abstract

Tumor cells often employ many ways to restrain type I interferon signaling to evade immune surveillance. However, whether cellular amino acid metabolism regulate this process remains unclear and its effects on antitumor immunity are relatively unexplored. Here, we find that asparagine inhibits IFN-I signaling and promotes immune escape in bladder cancer. Depletion of asparagine synthetase (ASNS) strongly limits in vivo tumor growth in a CD8+ T cell-dependent manner and boosts immunotherapy efficacy. Moreover, clinically approved ASNase synergizes with anti-PD-1 therapy in suppressing tumor growth. Mechanistically, asparagine can directly bind to RIG-I and facilitate CBL-mediated RIG-I degradation, thereby suppressing IFN signaling and antitumor immune responses. Clinically, tumors with higher ASNS expression show decreased responsiveness to ICIs therapy. Together, our findings uncover asparagine as a natural metabolite to modulate RIG-I-mediated IFN-I signaling, providing the basis for developing the combinatorial use of ASNase and anti-PD-1 for bladder cancer.

Authors

Wenjie Wei, Hongzhao Li, Shuo Tian, Chi Zhang, Junxiao Liu, Wen Tao, Tianwei Cai, Yuhao Dong, Chuang Wang, Dingyi Lu, Yakun Ai, Wanlin Zhang, Hanfeng Wang, Kan Liu, Yang Fan, Yu Gao, Qingbo Huang, Xin Ma, Baojun Wang, Xu Zhang, Yan Huang