We describe a previously-unappreciated role for Bruton's tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B-cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, BTKi-treated patients, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in susceptible patients.
Jigar V. Desai, Marissa A. Zarakas, Andrew L. Wishart, Mark Roschewski, Mariano A. Aufiero, Ágnes Donkó, Gustaf Wigerblad, Neta Shlezinger, Markus Plate, Matthew R. James, Jean K. Lim, Gulbu Uzel, Jenna R.E. Bergerson, Ivan Fuss, Robert A. Cramer, Luis M. Franco, Emily S. Clark, Wasif N. Khan, Daisuke Yamanaka, Georgios Chamilos, Jamel El-Benna, Mariana J. Kaplan, Louis M. Staudt, Thomas L. Leto, Steven M. Holland, Wyndham H. Wilson, Tobias M. Hohl, Michail S. Lionakis
BACKGROUND. Features of consumptive coagulopathy and thromboinflammation are prominent in cerebral malaria (CM). We hypothesized that thrombogenic autoantibodies contribute to a procoagulant state in CM. METHODS. Plasma from children with uncomplicated malaria (UM, n = 124) and CM (n = 136) was analyzed by ELISA for a panel of 8 autoantibodies including anti-Platelet Factor 4/polyanion (anti-PF4/P), anti-Phospholipid, anti-Phosphatidylserine, anti-Myeloperoxidase, anti-Proteinase 3, anti-dsDNA, anti-Beta-2-Glycoprotein I (β2GPI), and anti-Cardiolipin. Non-malaria coma (NMC, n = 49) and healthy controls (HC, n = 56) were assayed for comparison. Associations with clinical and immune biomarkers were determined using univariate and logistic regression analyses. RESULTS. Median anti-PF4/P and anti-PS IgG levels were elevated with malaria infection relative to HC (P < 0.001) and NMC (PF4/P: P < 0.001). Anti-PF4/P IgG levels were elevated in CM (median = 0.27, IQR: 0.19–0.41) compared to UM (median = 0.19, IQR: 0.14–0.22, P ≤ 0.0001). Anti-PS IgG levels did not differ between UM and CM (P = 0.39). When CM cases were stratified by malaria retinopathy (Ret) status, levels of anti-PF4/P IgG correlated negatively with peripheral platelet count in Ret+ CM cases (Rs = 0.201, P = 0.04) and associated positively with mortality (OR = 15.2, 95% CI: 1.02–275, P = 0.048). Plasma from CM patients induced a greater platelet activation capacity in an ex-vivo assay relative to plasma from UM patients (P = 0.02). Platelet activation was associated with anti-PF4/P IgG levels (Rs = 0.293, P = 0.035). CONCLUSIONS. Thrombosis mediated by elevated anti-PF4/P autoantibodies may be one mechanism contributing to the clinical complications of CM.
Iset M. Vera, Anne Kessler, Visopo Harawa, Ajisa Ahmadu, Thomas E. Keller, Stephen T.J. Ray, Terrie E. Taylor, Stephen J. Rogerson, Wilson L. Mandala, Morayma Reyes Gil, Karl B. Seydel, Kami Kim
Individuals with clonal hematopoiesis of indeterminate potential (CHIP) are at increased risk of aging related health conditions and all-cause mortality, but whether CHIP impacts risk of infection is much less clear. Using UK Biobank data, we revealed a positive association between CHIP and incident pneumonia in 438,421 individuals. We show that inflammation enhanced pneumonia risk, as CHIP carriers with a hypomorphic IL6 receptor polymorphism were protected. To better characterize the pathways of susceptibility, we challenged hematopoietic Tet Methylcytosine Dioxygenase 2 knockout (Tet2–/–) and floxed control mice (Tet2f/f) with Streptococcus pneumoniae. As with human CHIP carriers, Tet2–/– mice had hematopoietic abnormalities resulting in the expansion of inflammatory monocytes and neutrophils in peripheral blood. Yet, these cells were insufficient in defending against S. pneumoniae and resulted in increased pathology, impaired bacterial clearance, and higher mortality in Tet2–/– mice. We delineated the transcriptional landscape of Tet2–/– neutrophils and found that while inflammation-related pathways were upregulated in Tet2–/– neutrophils, migration and motility pathways were compromised. Using live-imaging techniques, we demonstrated impairments in motility, pathogen uptake and neutrophil extracellular trap (NET) formation by Tet2–/– neutrophils. Collectively, we show that CHIP is a risk factor for bacterial pneumonia related to innate immune impairments.
Candice Quin, Erica N. DeJong, Elina K. Cook, Yi Zhen Luo, Caitlyn Vlasschaert, Sanathan Sadh, Amy J.M. McNaughton, Marco M. Buttigieg, Jessica A Breznik, Allison E. Kennedy, Kevin Zhao, Jeffrey Mewburn, Kimberly J. Dunham-Snary, Charles C.T. Hindmarch, Alexander G. Bick, Stephen L. Archer, Michael J. Rauh, Dawn M.E. Bowdish
Staphylococcus aureus is a leading cause of biofilm-associated prosthetic joint infection (PJI). A primary contributor to infection chronicity is an expansion of granulocytic myeloid-derived suppressor cells (G-MDSCs) that are critical for orchestrating the anti-inflammatory biofilm milieu. Single-cell sequencing and bioinformatic metabolic algorithms were used to explore the link between G-MDSC metabolism and S. aureus PJI outcome. Glycolysis and the hypoxia response through hypoxia-inducible factor-1 alpha (HIF-1a) were significantly enriched in G-MDSCs. Interfering with both pathways in vivo, using a 2-deoxyglucose nanopreparation and granulocyte-targeted HIF-1a conditional knockout mice, respectively, attenuated G-MDSC-mediated immunosuppression and reduced bacterial burden in a mouse model of S. aureus PJI. In addition, scRNA-seq analysis of granulocytes from PJI patients also showed an enrichment in glycolysis and hypoxia response genes. These findings support the importance of a glycolysis/HIF-1a axis in promoting G-MDSC anti-inflammatory activity and biofilm persistence during PJI.
Christopher M. Horn, Prabhakar Arumugam, Zachary Van Roy, Cortney E. Heim, Rachel W. Fallet, Blake P. Bertrand, Dhananjay Shinde, Vinai C. Thomas, Svetlana G. Romanova, Tatiana K. Bronich, Curtis W. Hartman, Kevin L. Garvin, Tammy Kielian
BACKGROUND. Malaria transmission blocking vaccines aim to interrupt the transmission of malaria from one person to another. METHODS. The candidates, R0.6C and ProC6C, share the Plasmodium falciparum sexual stage antigen, Pfs48/45 “6C” domain. R0.6C utilizes the Glutamate Rich Protein (GLURP) as a carrier and ProC6C includes a second domain (Pfs230-Pro) and a short 36 amino acids CSP sequence. Healthy adults (n = 125) from a malaria endemic area of Burkina Faso were immunized with three intramuscular injections, four weeks apart, of 30 μg or 100 μg R0.6C or ProC6C each adsorbed to Alhydrogel adjuvant (AlOH) alone or in combination with Matrix-M (15 μg or 50 μg, respectively). The allocation was random and double blind for this Phase 1 trial. RESULTS. The vaccines were safe and well tolerated with no vaccine-related serious adverse events. A total of seven adverse events, mild to moderate in intensity and considered possibly related to the study vaccines were recorded. Vaccine-specific antibodies were highest in volunteers immunized with 100 μg ProC6C-AlOH with Matrix-M, and 13/20 (65%) subjects in the group showed greater than 80% transmission reducing activity (TRA) when evaluated in the standard membrane feeding assay at 15 mg/mL IgG. In contrast, R0.6C induced sporadic TRA. CONCLUSIONS. All formulations were safe and well tolerated in a malaria endemic area of Africa in healthy adults. The ProC6C-AlOH/Matrix-M vaccine elicited the highest levels of functional antibodies, meriting further investigation. TRIAL REGISTRATION. Pactr.org PACTR202201848463189. FUNDING. The study was funded by the European Union and Developing Countries Clinical Trials Partnership (Grant number RIA2018SV-2311).
B. Alfred Tiono, Jordan L. Plieskatt, Alphonse Ouedraogo, Ben Idriss Soulama, Kazutoyo Miura, Edith C. Bougouma, Mohammad Naghizadeh, Aissata Barry, Jean Baptiste B. Yaro, Sem Ezinmegnon, Noelie B. Henry, Ebenezer Ofori, Bright Adu, Susheel K. Singh, Augustin Konkobo, Karin Lövgren Bengtsson, Amidou Diarra, Cecilia Carnrot, Jenny M. Reimer, Amidou Z. Ouedraogo, Moussa Tienta, Carole A. Long, Issa N. Nebie, Issaka Sagara, Sodiomon B. Sirima, Michael Theisen
BACKGROUND. Vaccination is typically administered without regard to site of prior vaccination but this factor may substantially impact downstream immune responses. METHODS. We assessed serological responses to initial COVID-19 vaccination in baseline seronegative adults who received second–dose boosters in the ipsilateral or contralateral arm relative to initial vaccination. We measured serum SARS-CoV2 spike-specific Ig, RBD-specific IgG, SARS-CoV-2-nucleocapsid-specific IgG, and neutralizing antibody titers against SARS-CoV-2.D614G (early strain) and SARS-CoV-2.B.1.1.529 (Omicron) at approximately 0.6, 8, and 14 months after boosting. RESULTS. In 947 individuals, contralateral boosting was associated with higher spike-specific serum Ig, and this effect increased over time from a 1.1-fold to a 1.4-fold increase by 14 months (P < 0.001). A similar pattern was seen for RBD-specific IgG. Among 54 pairs matched for age, gender and relevant time intervals, arm groups had similar antibody levels at W2 but contralateral boosting resulted in significantly higher binding and neutralizing antibody titers at W3 and W4, with progressive increase over time, ranging from 1.3-fold (total Ig, P = 0.007) to 4.0-fold (pseudovirus neutralization to B.1.1.529 P < 0.001). CONCLUSIONS. In previously unexposed adults receiving an initial vaccine series with the BNT162b2 mRNA COVID-19 vaccine, contralateral boosting substantially increases antibody magnitude and breadth at times beyond 3 weeks after vaccination. This effect should be considered during arm selection in the context of multi-dose vaccine regimens.
Sedigheh Fazli, Archana Thomas, Abram E. Estrada, Hiro A.P. Ross, David Xthona Lee, Steven Kazmierczak, Mark K. Slifka, David Montefiori, William B. Messer, Marcel E. Curlin
BACKGROUND. Sanaria PfSPZ Vaccine, composed of attenuated Plasmodium falciparum (Pf) sporozoites (SPZ), protects against malaria. We conducted this clinical trial to assess the safety and efficacy of PfSPZ Vaccine in HIV positive (HIV+) individuals since the HIV infection status of participants in mass vaccination programs may be unknown. METHODS. This randomized, double blind, placebo-controlled trial enrolled 18-45-year-old HIV negative (HIV-) and well-controlled HIV+ Tanzanians (HIV viral load < 40 copies/mL, CD4 counts > 500 cells/µL). Participants received 5 doses of PfSPZ Vaccine or normal saline over 28 days followed by controlled human malaria infection (CHMI) 3 weeks later. RESULTS. There were no solicited adverse events in the 9 HIV- and 12 HIV+ participants. After CHMI, 6/6 normal saline (NS) controls, 1/5 HIV- vaccinees and 4/4 HIV+ vaccinees were Pf positive by qPCR. Post-immunization, anti-PfCSP (isotype and IgG subclass) and anti-PfSPZ antibodies, anti-PfSPZ CD4 T cell responses and Vδ2+ γδ CD3+ T cells were non-significantly higher in HIV- than HIV+ vaccinees. Sera from HIV- vaccinees had significantly higher inhibition of PfSPZ invasion of hepatocytes in vitro, and antibody-dependent complement deposition (ADCD) and Fcγ3B binding by anti-PfCSP and ADCD by anti-PfCelTOS antibodies. CONCLUSIONS. PfSPZ Vaccine was safe and well tolerated in HIV+ vaccinees, but not protective. Vaccine efficacy was 80% in HIV- vaccinees (P = 0.012), whose sera had significantly higher inhibition of PfSPZ invasion of hepatocytes and enrichment of multi-functional PfCSP antibodies. A more potent PfSPZ vaccine or regimen is needed to protect those living with HIV against Pf infection in Africa.
Said Jongo, L.W. Preston Church, Florence Milando, Munira Qassim, Tobias Schindler, Mohammed Rashid, Anneth Tumbo, Gloria Nyaulingo, Bakari M. Bakari, Thabit Athuman Mbaga, Latipha Mohamed, Kamaka Kassimu, Beatus S. Simon, Maxmillian Mpina, Irfan Zaidi, Patrick E. Duffy, Phillip A. Swanson II, Robert Seder, Jonathan D. Herman, Maanasa Mendu, Yonatan Zur, Galit Alter, Natasha KC, Pouria Riyahi, Yonas Abebe, Tooba Murshedkar, Eric R. James, Peter F. Billingsley, B. Kim Lee Sim, Thomas L. Richie, Claudia Daubenberger, Salim Abdulla, Stephen L. Hoffman
Fang Yun Lim, Soo-Young Kim, Karisma N. Kulkarni, Rachel L. Blazevic, Louise E. Kimball, Hannah G. Lea, Amanda J. Haack, Maia S. Gower, Terry Stevens-Ayers, Lea M. Starita, Michael Boeckh, Ollivier Hyrien, Joshua T. Schiffer, Ashleigh B. Theberge, Alpana Waghmare
BACKGROUND. Disease due to dengue viruses is a growing global health threat, causing 100-400 million cases annually. An ideal dengue vaccine should demonstrate durable protection against all four serotypes in phase 3 efficacy trials, however the lack of circulating serotypes may lead to incomplete efficacy data. Controlled human infection models help down select vaccine candidates and supply critical data to supplement efficacy trials. We evaluated the efficacy of a leading live attenuated tetravalent dengue vaccine candidate, TV005, against infection with a newly established dengue serotype 3 or an established serotype 2 challenge virus. METHODS. Two randomized controlled clinical trials were performed. In study 1, 42 subjects received TV005 or placebo (n = 21 each) and six months later all were challenged with dengue 2 virus (DEN2Δ30) at a dose of 103 PFU. In study 2, 23 subjects received TV005, 20 subjects received placebo and six months later all were challenged with 104 PFU dengue 3 virus (DEN3Δ30). Subjects were closely monitored for safety, viremia, and immunologic responses. Infection, measured by post-challenge viremia and by occurrence of rash and neutropenia, were the primary endpoints. Secondary endpoints included safety, immunologic, and virologic profiles following vaccination with TV005 and subsequent DENV2 or DENV3 challenge strains. RESULTS. TV005 was well tolerated and protected all vaccinated volunteers from viremia with DENV2 or 3 (none infected in either group). Placebo recipients had viremia post-challenge (100% in study 1, 85% in study 2) and all experienced rash following challenge with either serotype. CONCLUSIONS. TV005 is a leading tetravalent dengue vaccine candidate which fully protects against infection with DENV2 and DENV3 in an established controlled human infection model. CLINICAL TRIALS REGISTRATION NUMBERS. NCT02317900 and NCT02873260.
Kristen K. Pierce, Anna P. Durbin, Mary-Claire Walsh, Marya Carmolli, Beulah P. Sabundayo, Dorothy M. Dickson, Sean A. Diehl, Stephen S. Whitehead, Beth D. Kirkpatrick
Ruangang Pan, David K. Meyerholz, Stanley Perlman
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