T cell immunity is essential for the control of tuberculosis (TB), an important disease of the lung, and is generally studied in humans using peripheral blood cells. Mounting evidence, however, indicates that tissue resident memory T cells (Trm) are superior at controlling many pathogens, including Mycobacterium tuberculosis (Mtb), and can be quite different from those in circulation. Using freshly resected lung tissue, from individuals with active or previous TB, we identified distinct CD4 and CD8 Trm-like clusters within TB diseased lung tissue that were functional and enriched for IL-17 producing cells. Mtb-specific CD4 T cells producing TNF-α, IL-2 and IL-17 were highly expanded in the lung compared to matched blood samples, in which IL-17+ cells were largely absent. Strikingly, the frequency of Mtb-specific lung T cells making IL-17, but not other cytokines, inversely correlated with the plasma IL-1β levels, suggesting a potential link with disease severity. Using a human granuloma model, we showed the addition of either exogenous IL-17 or IL-2 enhanced immune control of Mtb and was associated with increased NO production. Taken together, these data support an important role for Mtb-specific Trm-like IL-17 producing cells in the immune control of Mtb in the human lung.
Paul Ogongo, Liku B. Tezera, Amanda Ardain, Shepherd Nhamoyebonde, Duran Ramsuran, Alveera Singh, Abigail Ngoepe, Farina Karim, Taryn Naidoo, Khadija Khan, Kaylesh J. Dullabh, Michael Fehlings, Boon Heng Lee, Alessandra Nardin, Cecilia S. Lindestam Arlehamn, Alessandro Sette, Samuel M. Behar, Adrie J.C. Steyn, Rajhmun Madansein, Henrik N. Kløverpris, Paul T. Elkington, Alasdair Leslie
Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS-CoV-2 viral entry and may represent such a target. We hypothesized that drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool named 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo. Several candidate compounds, Avoralstat, PCI-27483, Antipain, and Soybean-Trypsin-Inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays. Avoralstat, a clinically tested Kallikrein-related B1 inhibitor, inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle, Avoralstat significantly reduced lung tissue titers and mitigated weight-loss when administered prophylactically to SARS-CoV-2 susceptible mice indicating its potential to be repositioned for COVID-19 prophylaxis in humans.
Young Joo Sun, Gabriel Velez, Dylan E. Parsons, Kun Li, Miguel E. Ortiz, Shaunik Sharma, Paul B. McCray Jr., Alexander G. Bassuk, Vinit B. Mahajan
Tissue-based T cells are important effectors in the prevention and control of mucosal viral infections – less is known about tissue-based B cells. We demonstrate that B cells and antibody-secreting cells (ASCs) are present in inflammatory infiltrates in skin biopsies of persons during symptomatic HSV2 reactivation and early healing. Both CD20+ B cells, most of which are antigen-inexperienced by co-expression of IgD, and ASCs, characterized by dense IgG RNA expression in combination with CD138, IRF4 and Blimp1 RNA, are seen in association with T cells. ASCs are found clustered with CD4+ T cells, suggesting potential for crosstalk. HSV2-specific antibodies to virus surface antigens are also present in tissue and increase in concentration during HSV2 reactivation and healing, unlike in serum where concentrations remain static over time. B cells, ASCs, and HSV-specific antibody were rarely detected in biopsies of unaffected skin. Evaluation of serial biopsies demonstrate that B cells and ASCs follow a more migratory than resident pattern of infiltration in HSV-affected genital skin, in contrast to T cells. Together, these observations suggest distinct phenotypes of B cells in HSV-affected tissue; dissecting their role in reactivation may reveal new therapeutic avenues to control these infections.
Emily S. Ford, Anton M. Sholukh, RuthMabel Boytz, Savanna S. Carmack, Alexis Klock, Khamsone Phasouk, Danica Shao, Raabya Rossenkhan, Paul T. Edlefsen, Tao Peng, Christine Johnston, Anna Wald, Jia Zhu, Lawrence Corey
A(H3N2) Influenza vaccine effectiveness (VE) were low during 2016-2019 seasons and varied by age. We analyzed neutralizing antibody responses to egg- and cell-propagated vaccine and circulating viruses following vaccination in 375 individuals (aged 7 months to 82 years) across all vaccine eligible age groups in 3 influenza seasons. Antibody responses to cell- compared to egg-propagated vaccine viruses were significantly reduced due to egg-adapted changes T160K, D225G, and L194P in the vaccine hemagglutinins. Vaccine egg-adaptation had differential impact on antibody responses across different age groups. Immunologically naive children immunized with egg-adapted vaccines mostly mounted antibodies targeting egg-adapted epitopes, whereas those previously primed with infection produced broader responses even when vaccinated with egg-based vaccines. In elderly, repeated boost of vaccine egg-adapted epitopes significantly reduced antibody responses to the wild type cell-grown viruses. Analysis with reverse genetics viruses suggested that the response to each egg-adapted substitution varied by age. Antibody responses did not differ in male versus female vaccinees. Here, the combination of age-specific responses to vaccine egg-adapted substitutions, diverse host immune priming histories and virus antigenic drift impacted antibody responses following vaccination and may have led to the low and variable VE against A(H3N2) viruses across different age groups.
Feng Liu, F. Liaini Gross, Stacie N. Jefferson, Crystal Holiday, Yaohui Bai, Li Wang, Bin Zhou, Min Z. Levine
COVID-19 convalescent plasma, particularly plasma with high-titer SARS-CoV-2 (CoV2) antibodies, is one of the leading treatments for individuals with early COVID-19 infection. The functionality of convalescent plasma varies greatly, but the association of antibody epitope specificities with plasma functionality remains uncharacterized. We assessed antibody functionality and reactivities to peptides across the CoV2 and the four endemic human coronavirus (HCoV) genomes in 126 COVID-19 convalescent plasma donations. We found strong correlation between plasma functionality and polyclonal antibody targeting of CoV2 spike protein peptides. Antibody reactivity to many HCoV spike peptides also displayed strong correlation with plasma functionality, including pan-coronavirus cross-reactive epitopes located in a conserved region of the fusion peptide. After accounting for antibody cross-reactivity, we identified an association between greater alphacoronavirus NL63 antibody responses and development of highly neutralizing antibodies to SARS-CoV-2. We also found that plasma preferentially reactive to the CoV2 spike receptor binding domain (RBD), versus the betacoronavirus HKU1 RBD, had higher neutralizing titer. Finally, we developed a two-peptide serosignature that identifies plasma donations with high anti-spike titer, but that suffer from low neutralizing activity. These results suggest that analysis of coronavirus antibody fine specificities may be useful for selecting therapeutic plasma with desired functionalities.
William R. Morgenlander, Stephanie N. Henson, Daniel R. Monaco, Athena Chen, Kirsten Littlefield, Evan M. Bloch, Eric Fujimura, Ingo Ruczinski, Andrew R. Crowley, Harini Natarajan, Savannah E. Butler, Joshua A. Weiner, Mamie Z. Li, Tania S. Bonny, Sarah E. Benner, Ashwin Balagopal, David Sullivan, Shmuel Shoham, Thomas C. Quinn, Susan Eshleman, Arturo Casadevall, Andrew D. Redd, Oliver Laeyendecker, Margaret E. Ackerman, Andrew Pekosz, Stephen J. Elledge, Matthew L. Robinson, Aaron A.R. Tobian, H. Benjamin Larman
Multiple studies have shown loss of SARS-CoV-2 specific antibodies over time after infection, raising concern that humoral immunity against the virus is not durable. If immunity wanes quickly, millions of people may be at risk for reinfection after recovery from COVID-19. However, memory B cells (MBC) could provide durable humoral immunity even if serum neutralizing antibody titers decline. We performed multi-dimensional flow cytometric analysis of S protein receptor binding domain (S-RBD)-specific MBC in cohorts of ambulatory COVID-19 patients with mild disease (n = 7), and hospitalized patients with moderate to severe disease (n = 7), at a median of 54 (39-104) days after symptom onset. We detected S-RBD-specific class-switched MBC in 13 of 14 participants, failing only in the individual with lowest plasma levels of anti-S-RBD IgG and neutralizing antibodies. Resting MBC (rMBC) made up the largest proportion of S-RBD-specific MBC in both cohorts. FCRL5, a marker of functional memory on rMBC, was more dramatically upregulated on S-RBD-specific rMBC after mild infection than after severe infection. These data indicate that most SARS-CoV-2-infected individuals develop S-RBD-specific, class-switched rMBC that resemble germinal center-derived B cells induced by effective vaccination against other pathogens, providing evidence for durable B cell-mediated immunity against SARS-CoV-2 after mild or severe disease.
Clinton O. Ogega, Nicole E. Skinner, Paul W. Blair, Han-Sol Park, Kirsten Littlefield, Abhinaya Ganesan, Santosh Dhakal, Pranay Ladiwala, Annukka A.R. Antar, Stuart C. Ray, Michael J. Betenbaugh, Andrew Pekosz, Sabra L. Klein, Yukari C. Manabe, Andrea L. Cox, Justin R. Bailey
Background. Vaccines that block human-to-mosquito Plasmodium transmission are needed for malaria eradication and clinical trials have targeted zygote antigen Pfs25 for decades. We reported that a Pfs25 protein-protein conjugate vaccine formulated in alum adjuvant induced significant serum functional activity in both US and Malian adults. However, antibody titers declined rapidly, and transmission-reducing activity required four vaccine doses. Functional immunogenicity and durability must be improved before advancing TBV further in clinical development. We hypothesized that the pre-fertilization protein Pfs230 alone or in combination with Pfs25 would improve functional activity.Methods. Transmission-blocking vaccine candidates based on gamete antigen Pfs230 or Pfs25 were conjugated with Exoprotein A, formulated in Alhydrogel, and administered to mice, rhesus macaques, and humans. Antibody titers were measured by ELISA and transmission-reducing activity was assess by the Standard Membrane Feeding Assay. Results. Pfs25-EPA/Alhydrogel and Pfs230D1-EPA/Alhydrogel induced similar serum functional activity in mice, but Pfs230D1-EPA induced significantly greater activity in rhesus monkeys that was enhanced by complement. In U.S. adults, two vaccine doses induced complement-dependent activity in 4 of 5 Pfs230D1-EPA/Alhydrogel recipients but no significant activity in five Pfs25-EPA recipients, and combination with Pfs25-EPA did not increase activity over Pfs230D1-EPA alone.Conclusion. The complement-dependent functional immunogenicity of Pfs230D1-EPA represents a significant improvement over Pfs25-EPA in this comparative study. The rhesus model is more predictive of the functional human immune response to Pfs230D1 than is the mouse model. Trial Registration. ClinicalTrials.gov NCT02334462Funding. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Sara A. Healy, Charles F. Anderson, Bruce J. Swihart, Agnes Mwakingwe-Omari, Erin E. Gabriel, Hope Decederfelt, Charlotte V. Hobbs, Kelly M. Rausch, Daming Zhu, Olga Muratova, Raul Herrera, Puthupparampil V. Scaria, Nicholas J. MacDonald, Lynn E. Lambert, Irfan Zaidi, Camila H. Coelho, Jonathan P. Renn, Yimin Wu, David L. Narum, Patrick E. Duffy
Group B Streptococcus (GBS) is the major cause of human neonatal infections. A single clone, designated CC17-GBS, accounts for more than 80% of meningitis cases, the most severe form of the infection. However, the events allowing blood-borne GBS to penetrate the brain remain largely elusive. In this study, we identified the host transmembrane receptors α5β1 and αvβ3 integrins as the ligands of Srr2, a major CC17-GBS specific adhesin. Two motifs located in the binding region of Srr2 were responsible for the interaction between CC17-GBS and these integrins. We demonstrated, in a blood-brain barrier cellular model, that both integrins contributed to the adhesion and internalization of CC17-GBS. Strikingly, both integrins were overexpressed during the post-natal period in the brain vessels of the blood-brain and blood-cerebrospinal fluid barriers and contributed to the juvenile susceptibility to CC17-meningitis. Finally, blocking these integrins decreased CC17-GBS crossing into the juvenile mice central nervous system in an in vivo model of meningitis.Our study demonstrates that CC17-GBS exploits integrins for crossing the brain vessels leading to meningitis. Importantly, it provides host molecular insights into neonate’s susceptibility to CC17-GBS meningitis, thereby opening new perspectives for therapeutic and prevention strategies of GBS-elicited meningitis.
Romain Deshayes de Cambronne, Agnès Fouet, Amandine Picart, Anne-Sophie Bourrel, Cyril Anjou, Guillaume Bouvier, Cristina Candeias, Abdelouhab Bouaboud, Lionel Costa, Anne-Cécile Boulay, Martine Cohen-Salmon, Isabelle Plu, Caroline Rambaud, Eva Faurobert, Corinne Albiges-Rizo, Asmaa Tazi, Claire Poyart, Julie Guignot
Early appearance of neutralizing antibodies during acute hepatitis C virus (HCV) infection is associated with spontaneous viral clearance. However, the longitudinal changes in antigen-specific memory B cell (MBCs) associated with divergent HCV infection outcomes remain undefined. We characterized longitudinal changes in E2 glycoprotein-specific MBCs from subjects who either spontaneously resolved acute HCV infection or progressed to chronic infection, using single-cell RNA-seq and functional assays. HCV-specific antibodies in plasma from chronically infected subjects recognized multiple E2 genotypes, while those from spontaneous resolvers exhibited variable cross-reactivity to heterotypic E2. E2-specific MBCs from spontaneous resolvers peaked early after infection (4–6 months), following expansion of activated circulating T follicular helper cells (cTfh) expressing interleukin 21. In contrast, E2-specific MBCs from chronically infected subjects, enriched in VH1-69, expanded during persistent infection (> 1 year), in the absence of significantly activated cTfh expansion. Early E2-specific MBCs from spontaneous resolvers produced monoclonal antibodies (mAbs) with fewer somatic hypermutations and lower E2 binding but similar neutralization as mAbs from late E2-specific MBCs of chronically infected subjects. These findings indicate that early cTfh activity accelerates expansion of E2-specific MBCs during acute infection, which might contribute to spontaneous clearance of HCV.
Eduardo Salinas, Maude Boisvert, Amit A. Upadhyay, Nathalie Bédard, Sydney A. Nelson, Julie Bruneau, Cynthia A. Derdeyn, Joseph Marcotrigiano, Matthew J. Evans, Steven E. Bosinger, Naglaa H. Shoukry, Arash Grakoui
Lymphatic filariasis is the major global cause of non-hereditary lymphoedema. We demonstrate the filarial nematode, Brugia malayi, induces lymphatic remodelling and impaired lymphatic drainage following parasitism of limb lymphatics in a mouse model. Lymphatic insufficiency was associated with elevated circulating lymphangiogenic mediators, including vascular endothelial growth factor C. Lymphatic insufficiency was dependent on type-2 adaptive immunity, interleukin-4 receptor, recruitment of C-C chemokine receptor-2 monocytes and alternatively-activated macrophages with pro-lymphangiogenic phenotype. Oral treatments with second-generation tetracyclines improved lymphatic function, while other classes of antibiotic had no significant effect. Second-generation tetracyclines directly targeted lymphatic endothelial cell proliferation and modified type-2 pro-lymphangiogenic macrophage development. Doxycycline treatment impeded monocyte recruitment, inhibited polarisation of alternatively-activated macrophages and suppressed T cell adaptive immune responses following infection. Our results determine a mechanism-of-action for the anti-morbidity effects of doxycycline in filariasis and supports clinical evaluation of second-generation tetracyclines as affordable, safe therapeutics for lymphoedemas of chronic inflammatory origin.
Julio Furlong-Silva, Stephen D. Cross, Amy E. Marriott, Nicolas Pionnier, John Archer, Andrew Steven, Stefan Schulte-Merker, Matthias Mack, Young-Kwon Hong, Mark J. Taylor, Joseph D. Turner
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