bluuebunny/crossref_metadata_2025 huggingface dataset:
CD206+MHCII− macrophages present in the neonatal but not adult intestine do not derive from LysM monocytes and are decreased in experimental Necrotizing Enterocolitis
Authors: Managlia Elizabeth , Yan Xiaocai , De Plaen Isabelle G
Published: January 2023

Necrotizing enterocolitis (NEC) is an intestinal disease targeting premature infants. While not well understood, its pathogenesis involves an underdeveloped intestinal immune system. In the neonate, this system is primarily innate, with Cx3cr1+ intestinal macrophages (Mφ) being the predominant population. In the murine neonatal intestine, the Cx3cr1+ compartment contains embryonic self-renewing Mφ (Ly6C− CD64+ MHCII+ Tim4+) that are largely replaced by monocyte-derived-Mφ at weaning, while only a small population of Tim4+ cells persist in adults. However, how inflammation affects the neonatal intestinal Mφ compartment is not known. Using flow cytometry, we found the neonatal but not adult murine intestine to contain a population of Cx3cr1Hi CD64Hi MHCII− cells (30.62% ± 1.6 of Cx3cr1+ cells). 28.7% ± 1.8 of these cells express CD206, a known marker of embryonic origin in other tissues. While 34.4% ± 1.2 of total CD206+ cells expressed Tim4 in dam fed pups, it increased to 49.3% ± 0.51 p≤ 0.01 in pups exposed to experimental NEC for 24 hours. This CD206+ Tim4+ subset was mainly MHCII+. When Cx3cr1-expressing cells were depleted (Cx3cr1-Cre CSFR1-DTR mice), all Cx3cr1-expressing cells were depleted, including CD206+ MHCII−and+, but not Ly6C+ monocytes. In contrast, when LysM-Cre was used (LysM-Cre CSFR1-DTR mice), pups had decreased Ly6C+ monocytes but the CD206+ MHCII−and+ populations were not depleted. These results were confirmed by immunofluorescent staining for CD206 and IBA-1. Thus, we conclude that neonatal intestinal CD206+ MHCII−or+ cells do not derive from LysM+ monocytes. We speculate that CD206+ MHCII− cells acquire MHCII during NEC thus decreasing the CD206+ MHCII− pool.

Supported by NIH DK116568 (IDP)
Neonatal Antibiotic Exposure Alters Intestinal Macrophage Frequency and Polarization
Authors: Schill Ellen Merrick , Udayan Sreeram , Gaddipatti Shreya , John Vini , Barrios Bibiana E , Floyd Alexandria N , McDonald Keely G , Newberry Rodney D
Published: January 2023

More than 75% of premature infants are given antibiotics in the first week of life. This is lifesaving for neonates with serious bacterial infections but is associated with increased risks of necrotizing enterocolitis (NEC). Given the association of intestinal macrophages (Mϕ) in the pathogenesis in NEC, we characterized the effect of antibiotics on this cell type. Pups were exposed to ampicillin and neomycin via the dam from postnatal day 1 (P1) to P7/8. Mϕ were identified as CD45+ CX3CR1+ CD11b+ F480+ using flow cytometry. In the small intestine (SI) there was an increase in the percentage of Mϕ in antibiotic-exposed lamina propria, but no difference in the proportion of F480-myeloid cells to Mϕ. The percentage of Mϕ in exposed colonic lamina propria cells was unchanged, but the proportion of Mϕ to F480-myeloid cells was increased. This suggests that antibiotic exposure expands myeloid cells in both the SI and colon, but likely via different mechanisms. To further characterize the phenotype of the intestinal Mϕ, we assessed polarity by immunoassay of 13 Mϕ associated cytokines. There were no significant differences in the proximal small intestine cytokine expression between control and antibiotic-exposed tissue. In the distal SI, antibiotic exposed mice had higher levels of TGFβ and CCL2, M2 Mϕ markers. In the colon, control tissue had elevated M1 Mϕ markers compared to antibiotic-exposed mice. Antibiotic-exposed colon had significantly increased levels of TGFβ. These data demonstrate that antibiotic exposure alters Mϕ proportion and polarization. Further work will be necessary to determine how this initial shift away from pro-inflammatory polarization leaves antibiotic-exposed infants at risk for intestinal inflammatory diseases.

Supported by NIH T32DK077653
A dietary fiber limits autoimmune neuroinflammation by restricting Th1 activation, polarization, and migration
Authors: Fettig Naomi M , Robinson Hannah G , Allanach Jessica R , Simister Rachel L , Wang Elsie J , Jiayu Ye , Davis Katherine M , Seo Jung Hee , Gibson Deanna L , Crowe Sean A , Horwitz Marc S , Osborne Lisa C
Published: January 2023

Dietary fibers and their breakdown products have been shown to limit inflammation in multiple disease contexts. However, the term “dietary fiber” encompasses a biochemically diverse family of carbohydrates, and it remains unknown how different fiber sources influence immune cell function. To address this question, we assessed the immunomodulatory capacity of different fiber types in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). C57BL/6 mice were fed a control diet (5% cellulose) or diets enriched (30%) with resistant starch, inulin, pectin, or guar gum for 2 weeks prior to EAE induction. A diet rich in guar gum uniquely ameliorated the clinical, pathological, and immunological features of EAE. Limited central nervous system (CNS) infiltration of IFNγ+ CD4+ T cells (Th1s) in guar gum-fed mice could be attributed to T cell-intrinsic impairment Th1 activation and polarization. Nanostring nCounter transcriptional analyses revealed downregulation of migration-associated markers in CD4+ T cells isolated from guar gum/EAE mice, which was supported by reduced cell-surface expression of integrins involved in migration into the CNS. Functionally, this translated to limited migration of guar gum-derived encephalitogenic Th1s into the CNS upon adoptive transfer into control-fed recipients, which reduced incidence and severity of EAE. However, the 16S rRNA microbiome signature and short chain fatty acid profiles were not unique in comparison to other fiber-supplemented diets, indicating a novel mechanism of action from those previously ascribed in high fiber diet studies. These data confirm the individuality of dietary fibers and identify a novel immunomodulatory function of guar gum.

This project was supported by the endMS doctoral studentship program (Multiple Sclerosis Society of Canada) (NMF, JRA), the Canadian Institutes for Health Research (PJT-148909), and the Canada Research Chair program (LCO).
Controllable regulation of CD3ζ and MyD88 signaling in T cells expands and enhances the neoantigen-reactive repertoire resulting in effective antitumor activity
Authors: Liu Jing , Davila Eduardo
Published: January 2023

Background

Adoptive cell therapy (ACT) of tumor-reactive T cells including tumor infiltrating lymphocytes (TIL) represents one of the most promising forms of therapies for treating patients with solid tumors. However, durable responses are observed in a small subset of patients and in certain cancer types. Major hurdles to achieving durable responses include: 1) suboptimal TCR responses due to sparse and/or weakly immunogenic tumor antigen presentation, 2) development of dysfunctional or exhausted T cells, 3) low TIL persistence, and 4) tumor infiltration. We and others have demonstrated that activating MyD88 signaling in T cells strongly amplifies weak TCR responses and imparts T cells novel anti-tumor properties.

Hypothesis

TILs engineered with a novel fusion protein consisting of CD3ζ:MyD88 will activate MyD88 signals in a TCR-dependent manner resulting in enhanced activity and against a broader panel of neoantigens.

Method & Results

CD4 and CD8 TILs from a variety of human tumors engineered CD3ζ:MyD88 demonstrated: a) dramatically improved ability to expand in vitro resulting (generating 10–100X TILs than conventional TILs within a few weeks), b) persist for months in tissue culture, c) more polyfunctional cells, d) destruction of established melanoma tumors in vivo, e) enhanced responses against a panel of antigens with no overt autoimmunity.

Conclusions

CD3ζ:MyD88 represents a unique and universal approach to safely enhance T cell response to tumor antigens.
Evaluation of dry DURAClone antibody panels for the characterization of human PBMCs, enriched T cell fractions and anti-BCMA CAR-T cells
Authors: Bowers Rita , Grazia Giulia , Kapinsky Michael , Larbi Anis , Lacaille Vashti
Published: January 2023

CAR-T cells are becoming a more common therapeutic approach and are setting forth to targeting of hematological disorders other than CD19+ entities and also solid tumors. However, the immunological determinants of CAR-T efficacy still need comprehensive investigation from starting material to final product, covering different CAR constructs, transfection methods and targets.

DURAClone dry antibody panels were designed to overcome standardization issues and operator-dependent impact on accuracy and precision in multicentric flow cytometry studies. The aim of this study was to evaluate the utility of DURAClone dry antibody panels for the characterization of PBMCs, derived T cell-enriched fractions as well as anti-BCMA CAR-T cells (ProMab Inc.), including MOCK-transfected controls.

DURAClone dry antibody panels were used as pre-configured or extended with CAR-specific probes and further antibodies. Viable CD45+ leukocyte and CD3+ T cell count, leukocyte composition (CD3/CD19/CD56/CD14/CD16) and the T cell (CD3/CD4/CD8) maturation profile (CD45RA, CD197/CCR7, CD27, CD57) including expression of checkpoint markers (LAG-3, PD-1) were assessed.

Regardless of sample type, absolute counts and cellular composition were assessed using streamlined procedures. Typical staining patterns for T cell maturation profile were observed, revealing unchanged proportions for the different stages between PBMCs and enriched T cell fractions and strong skewing toward effector cell phenotypes with elevated expression of a checkpoint markers in case of CAR-T cells. Our experiments demonstrate the flexible utility of DURAClone dry antibody panels for the phenotypic characterization from starting material to final CAR-T cell product.
Influenza virus-like particle-based hybrid vaccine containing RBD induces immunity against influenza and SARS-CoV-2 viruses
Authors: Bommireddy Ramireddy , Stone Shannon , Bhatnagar Noopur , Kumari Pratima , Munoz Luis E , Oh Judy , Berry Jameson L , Jacobson Kristen M , Jafaar Lahcen , Naing Swe-Htet , Blackerby Allison N , Van der Gaag Tori , Wright Chloe N , Kim Ki-Hye , Lai Lilin , Pack Christopher D , Ramachandiran Sampath , Suthar Mehul S , Kang Sang-Moo , Kumar Mukesh , Selvaraj Periasamy
Published: January 2023

Several approaches have produced an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the influence of immunity against other vaccinations on the durability and efficacy of the immune response against SARS-CoV-2 is still unknown in settings where vaccines against other viruses need to be administered simultaneously. This will be an important factor in developing multivalent vaccines against seasonal viruses. We have developed a hybrid vaccine for SARS-CoV-2 and influenza viruses using influenza virus-like particles (VLP) incorporated with glycosylphosphatidylinositol (GPI)-anchored Spike RBD of SARS-CoV-2 fused to GM-CSF as an adjuvant. GPI-anchored fusion protein of GM-CSF and the SARS-CoV-2 S1 RBD was expressed in CHO-S cells, purified by immunoaffinity chromatography and incorporated onto influenza VLPs by protein transfer to make a hybrid VLP vaccine. The efficacy of the hybrid VLP vaccine was tested against both SARS-CoV-2 and influenza A/PR8 viruses in a mouse model. Our results show that the hybrid vaccine induced a strong antibody response and protected the mice from both influenza virus and mouse-adapted SARS-CoV-2 challenges, with vaccinated mice having less body weight loss and significantly lower lung viral titers compared to control mice. These results suggest that the hybrid vaccine is a promising candidate for preventing influenza A and SARS-CoV-2 infections.

This work was supported by NIH/NIAID (SBIR Contract# 75N93019C00017 Amendment to Pack/Ramachandiran), and Intel Corporation for the Intel COVID-19 Global Technology Response Initiative grant.
Comparison of the Immunogenicity of five COVID-19 vaccines in Sri Lanka
Authors: Jeewandara J M K Chandima , Aberathna Inoka Sepali , Danasekara Saubhagya , Gomes Laksiri , Fernando Suranga , Guruge Dinuka , Ranasinghe Thushali , Gunasekera Banuri , Kamaladasa Achala , Kuruppu Heshan , Somathilake Gayasha , Jayamali Jeewantha , Jayathilaka Deshni , Wijayatilake Helanka Dinesh Kumara , Pushpakumara Pradeep Darshana , Harvie Michael , Nimasha Thashmi , de Silva Shiromi Devika Grace , Wijayamuni Ruwan , Schimanski Lisa , Rijal Pramila , Tan Jack , Townsend Alain , Ogg Graham S , Malavige Gathsaurie Neelika
Published: January 2023

We assessed antibody responses 3 months post-vaccination in those who received mRNA-1273 (n=225), Sputnik V (n=128) or the first dose of Gam-COVID-Vac (n=184) and compared the results with previously reported data of Sinopharm and AZD1222 vaccinees. 99.5% of Moderna >94% of AZD1222 or Sputnik V, 72% to 76% of Gam-COVID-Vac (first dose) and 38.1% to 68.3% of Sinopharm vaccinees had ACE2 blocking antibodies above the positive threshold. The ACE2 blocking antibody levels were highest to lowest was Moderna > Sputnik V/AZD1222 (had equal levels)> first dose of Gam-COVID-Vac > Sinopharm. All Moderna recipients had antibodies above the positive threshold to the ancestral (WT), B.1.1.7, B.1.351.1 and 80% positivity rate for B.1.617.2. Positivity rates of Sputnik V vaccinees for WT and variants, were higher than AZD1222 vaccinees, while Sinopharm vaccinees had the lowest positivity rates (<16.7%). These findings highlight the need for further studies to understand the effects on clinical outcomes.

Allergy, Immunology and Cell Biology Unit, University of Sri Jayewardenepura; the NIH, USA (grant number 5U01AI151788-02), UK Medical Research Council and the Foreign and Commonwealth Office for support. T.K.T. is funded by the Townsend-Jeantet Charitable Trust (charity number 1011770) and the EPA Cephalosporin Early Career Researcher Fund. A.T. are funded by the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (grant no. 2018-I2M-2-002).
Alternative lineage B cells utilizing fatty acid oxidation predict response to third dose COVID vaccination in solid organ transplant recipients
Authors: Thompson Elizabeth A , Roznik Katerina , Karaba Andrew , Cascino Katherine , Dhakal Santosh , Sena Laura , Biavatti Luca , Abedon Aura T , Alejo Jennifer L , Klein Sabra L , Warren Daniel , Quin Caroline X , Mitchel Jonathan , Garonzik-Wang Jacqueline , Leone Robert , Boyarsky Brian , Segev Dorry L , Tobian Aaron A R , Werbel William , Cox Andrea L , Bailey Justin R
Published: January 2023

Solid organ transplant recipients (SOTRs) demonstrate reduced seroconversion and increased breakthrough infection rates following standard two dose mRNA vaccination against SARs-CoV-2. However, within a prospective cohort of SOTRs, the majority of patients (72%) developed a positive anti-Spike (S) IgG following a third vaccine dose, compared to only 15% following two doses. Those who failed to respond to vaccination uniformly received mycophenolate mofetil (MMF). To better understand mechanisms underlying divergent vaccine responses, both global and S-specific B cells were evaluated using flow cytometry to assess immunologic and metabolic phenotypes. Prior to the third dose, 76% of SOTRs demonstrated detectable S-specific B cells even though only 15% had positive anti-S IgG titers. However, B cells were skewed towards a non-class switched phenotype in SOTRs compared to healthy controls. Response to a third dose was predicted by expanded populations of germinal center (GC)-like B cells and CD11c+ alternative lineage B cells with upregulation of carnitine palmitoyltransferase 1a (CPT1a), the rate limiting enzyme for fatty acid oxidation (FAO), a preferred energy source of GC B cells. SOTRs receiving high dose MMF demonstrated significantly lower expression of CPT1a compared to healthy controls, indicating an energetic deficit associated with MMF and failure to respond. Further, in vitro treatment of B cells with MMF reduced the ability to oxidize fatty acids and induced an accumulation of intracellular lipid droplets. Together, these data define alternative lineage B cells present in those who respond to a third vaccine dose, outlining FAO as a metabolic pathway that could be targeted to improve vaccine responses.
Pan-coronavirus neutralizing S2 human monoclonal antibodies and utility of direct respiratory administration as combination therapy with S1 antibodies against SARS-CoV-2
Authors: Kobie James , Park Jun-Gyu , Sarkar Sanghita , Basu Madhubanti , Loos Andreas , Woo Jennifer , Ye Chengjin , Truong Vu , Bowen Richard , Walter Mark R , Martinez-Sobrido Luis , Piepenbrink Michael
Published: January 2023

To determine if pan-coronavirus neutralizing antibodies are induced by SARS-CoV-2 infection, human monoclonal antibodies (mAb) targeting the highly conserved S2 domain of the Spike glycoprotein were cloned from B cells isolated from convalescent patients with rationally designed SARS-CoV-2 stabilized S2 and SARS-CoV/SARS-CoV-2 S1S2 chimera recombinant protein probes. mAbs emerged capable of binding SARS-CoV-2, including the variants of concern (Beta, Gamma, Delta, and Omicron), SARS-CoV and MERS-CoV. The most potent neutralizing mAb with greatest breadth, 1249A8, recognizes all beta-coronaviruses tested including high affinity for SARS-CoV-2, SARS-CoV, and MERS-CoV. The 1249A8 lineage appears to have developed from a pre-existing OC43-specific memory B cell that was expanded as result of SARS-CoV-2 infection, and further expanded following COVID-19 vaccination. Treating K18 hACE2 transgenic mice with 1249A8 intraperitoneally prior to co-infection with SARS-CoV-2 Wa-1 and Beta VoC strains prevented weight loss and death, with viral titers below detectable levels by 4 days post-infection. 1249A8 delivered as a single 4mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2/Delta significantly protected them from weight loss, with therapeutic activity enhanced when combined with an S1-specific neutralizing mAb. As little as 2 mg/kg of 1249A8 i.n. 20 hours following infection with SARS-CoV/Urbani protected hamsters from weight loss. 1249A8 recognizes a highly conserved region near HR2 of the S2 domain of Spike. These results indicate that potent pan-coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can inform pan-coronavirus vaccine development.

Supported in-part by Aridis Pharmaceuticals and NIH (R01AI161175).
Persistence of immune memory to SARS-CoV-2 vaccine in lymphoid tissue
Authors: Davis-Porada Julia , Rybkina Ksenia , Poon Maya M L , Caron Daniel P , Jensen Isaac J , Kubota Masaru , Lam Nora , Lee Yoon Seung , Matsumoto Rei , Morrison-Colvin Rory , Szabo Peter A , Ural Basak B , Wells Steven B , Farber Donna L
Published: January 2023

Immunization with novel mRNA vaccines against SARS-CoV-2 administered worldwide protects against COVID-19 through the generation of neutralizing antibodies directed against the viral spike protein. While immune responses generated from vaccines have been evaluated in blood, the cellular stores of memory T and B cells are maintained in tissues. Using tissues from vaccinated, but previously uninfected organ donors, we surveyed lymphoid and barrier sites for the presence of SARS-CoV-2 vaccine specific memory B and T cells. Fluorescently labeled, full length Spike and RBD probes were used to identify vaccine specific memory B cells, while vaccine specific T cells were identified by expression of activation induced markers following stimulation with peptide pools spanning the entire spike protein. All donors were confirmed negative for anti-nucleocapsid antibodies and positive for anti-spike and anti-RBD antibodies via ELISA. Spike specific T cells were identified in the blood, spleen, lung, and lung associated lymph nodes (LLN), but not bone marrow (BM). In all tissues, these vaccine specific T cells were primarily CD4 and exhibited a mixed central- and effector-memory phenotype; in the LLN the majority (up to 55%) were T-follicular helper cells (CXCR5+/PD-1+). Spike and RBD specific memory B cells were detected in blood, lung, spleen, BM, and LLN. These vaccine specific B cells in the LLN specifically expressed the tissue residency marker CD69, but only a minority had features of germinal center B cells. These results indicate that memory B and T cells generated from mRNA vaccination localize primarily to lymphoid organs, including those draining the lung, which is important for understanding their longevity and protective capacity.

Supported by grants from NIH (U19 AI128949, PO1 AI106697, T32 GM007367)
Evaluation of the long-term anti-Spike immune response following a novel coronavirus vaccine (CORVax): electroporation of SARS-CoV-2 Spike plasmid DNA plus IL12 plasmid DNA
Authors: Jensen Shawn M , Afentoulis Michael , Wegmann Keith , Canton David A , Twitty Christopher G , Fox Bernard A
Published: January 2023

The global SARS-CoV-2 (CoV2) pandemic has focused efforts to generate effective vaccines that induce potent and persistent immunity. Recently, we described a novel vaccine approach using electroporation (EP) of a plasmid encoding a prefusion stabilized CoV2 spike protein (CORVax) plus plasmid Interleukin-12 (IL-12). IL-12 is an effective vaccine adjuvant that increases Th1 and Th2 antibodies in the serum. Here we examine the persistence of anti-Spike antibodies present in the serum of mice one year following CORVax vaccination.

C57BL/6 and BALB/c were vaccinated intramuscular (IM) and/or intradermal (ID) with a plasmid encoding the CoV2 spike protein with or without plasmid-encoded murine IL-12. Mice received plasmid EP immediately following injection. Splenocytes and serum were harvested at various time points to interrogate virus-specific cellular responses as well anti-spike antibody titers.

Anti-Spike IgG antibodies were elicited by EP of CORVax (IC50 = 1/2112), as well as EP of CORVax combined with IL-12 (IC50 = 1/4214) approximately 40 days after the booster vaccination. These anti-Spike IgG titers decayed over time but were still present 1 year after vaccination: CORVax (IC50 = 1/351 day 146, IC50 = 1/208 day 383); CORVax + IL12 (IC50 = 1/590 day 146, IC50 = 1/266 day 383).

Our data shows that EP of CORVax induces IgG responses to CoV2 Spike and the CoV2 Spike receptor binding domain. At one year following vaccination the anti-Spike IgG titers were higher in mice that received CORVax plus IL-12, however the rate at which the titers waned from their initial peak was comparable whether the mice received IL-12 or not. Additional studies are ongoing to determine whether the addition of IL-12 will enhance an anti-Spike memory response.

Generous support from The Chiles Foundation, Nancy Lematta, and the Providence Medical Foundation.
Efficacy of pediatric SARS-CoV-2 vaccines against high-dose B.1.617.2 challenge one year after vaccination of infant rhesus macaques
Authors: Milligan Emma C , Olstad Katherine , Cano Patricio , Munt Jenny , Lindesmith Lisa , Scobey Trevor , Mallory Michael , Edwards Darin , Carfi Andrea , Corbett Kizzmekia , Graham Barney S , Tomai Mark A , Iyer Smita S , Baric Ralph , Reader Rachel , Van Rompay Koen , Dittmer Dirk P , Permar Sallie , De Paris Kristina
Published: January 2023

To advance SARS-CoV-2 vaccines in infants younger than 5 years, we tested the efficacy of two SARS-CoV-2 vaccine platforms against challenge with the delta variant one year after immunization of infant rhesus macaques (RM).

Infant RMs (n=8/ group; 2 month-old) were immunized intramuscularly at weeks 0 and 4 with 30 mg stabilized prefusion SARS-CoV-2 S-2P spike (S) protein (Washington strain) encoded by mRNA encapsulated in lipid nanoparticles (mRNA-LNP) or 15 mg S protein mixed with 3M-052 in stable emulsion (Protein). At 1 year, vaccinated and age-matched unvaccinated RM (n=8) were challenged intranasally (106 pfu) and intratracheally (2×106 pfu) with B.1.617.2. Lung pathology was blindly assessed on day 7. Viral RNA copies of the N gene (vRNA) were measured by qPCR in nasal and pharyngeal swabs.

Severe lung pathology was observed in 7 of 8 controls compared to 1 of 8 or 0 of 8 RM in the mRNA-LNP or protein group, respectively. On days 2 and 4, vRNA copies/ml were significantly higher in pharyngeal swabs of control RM (day 2: 4.2p>

Supported by grants from NIH PO1 AI117915
Respiratory humoral and cellular immune responses following COVID-19 mRNA vaccination
Authors: Tang Jinyi , Zeng Cong , Cox Thomas M , Li Chaofan , Son Young Min , Cheon In Su , Behl Supriya , Justin Taylor J , Chakaraborty Rana , Johnson Aaron J , Shiavo Dante N , Utz James P , Reisenauer Janani S , Midthun David E , Mullon John J , Edell Eric S , Vassallo Robert S , Kern Ryan , Liu Shan-lu , Sun Jie
Published: January 2023

Vaccination is the key for controlling COVID-19 pandemic. SARS-CoV-2 mRNA vaccines have been demonstrated to induce robust and persistent humoral and cellular immunity in the circulation, but the vaccine-induced immune responses in the respiratory tract remain largely elusive. Here, we examined SARS-CoV-2 S-specific antibody, B and T cell immune responses in the bronchoalveolar lavage (BAL) fluid and blood from unvaccinated, COVID-19 vaccinees or COVID-19 convalescents that recovered from prior moderate to severe SARS-CoV-2 infection. We found that mRNA vaccination induced significant SARS-CoV-2 S-specific CD4+ and CD8+ T cell responses in the blood but not in the BAL. Similarly, mRNA vaccination failed to provoke detectable SARS-CoV-2 S RBD-specific B cell responses in the BAL despite marked RBD-specific B cells were observed in the blood of COVID-19 vaccinees. In contrast, robust SARS-CoV-2 S-specific B and T cell responses were present in the BAL of COVID-19 convalescents. Furthermore, significant neutralizing antibody responses were only observed in the BAL from convalescents but not vaccinees, while both COVID-19 vaccinee and convalescent groups mounted marked neutralizing antibody responses in the blood. Thus, despite their induction of robust circulating humoral and cellular immunity, the current COVID-19 vaccines provoke relatively weak cellular and neutralizing antibody responses in the lower respiratory tract. Our results indicate that a mucosal booster vaccine may be needed to establish robust immunity in the respiratory mucosa, which could provide a strong first line of defense against SARS-CoV-2 re-infection.
Development of a Cell-based ELISA assay to assess memory IgG produced in vitro by B cells specific for spike protein of SARS-CoV-2
Authors: Scapigliati Giuseppe , Zarletti Gianpaolo , Tiberi Massimo , De Molfetta Veronica , Toppi Elisa , Bossù Paola
Published: January 2023

Knowledge of antibody-mediated immune response to SARS-CoV-2 is crucial to understand the virus immunogenicity, establish seroprevalence and determine possible risks for infection/reinfection. An antibody presence, indicative of an humoral immune response, can be evidenced at early/intermediated stages after infection and antibodies are mainly detected by ELISA-based platforms. However, a major drawback of ELISA systems is a decrease of serum antibody titers during time. On the other hand, circulating antigen-specific memory B cells (MBC) may be present for long time after immunization/infection. The spike S1 protein has been shown to be a serological marker for Covid-19, and we describe a novel, simple and robust Cell-ELISA assay specifically designed to measure viral spike S1 protein by detecting specific IgG produced in vitro by MBC in PBMC (doi:10.3390/v12111274). By applying the Cell-ELISA assay to a cohort of 150 asympto/symptomatic individuals, age ranging from 8–100 years, we detected individuals resulted negative in ELISA but positive in Cell-ELISA, thus showing the presence of MBC that produce in vitro antibodies against SARS-CoV-2 at levels that are undetectable in the serum. These data may challenge the negative results obtained from the serological screening and indicate a previous antigen exposure in the possible absence of serum IgG. We also detected a presence of MBC specific for S1 protein for >10 months after viral infection (doi:10.3390/v13091704), suggesting a potent antigenicity, and evaluated the relative amount of in vitro secreted IgG in positive subjects. The proposed assay could be a candidate tool to monitor the presence of an effective IgG antibody memory after SARS-CoV-2 infection and vaccination.
Longitudinal measurement of human immune responses to SARS-CoV-2 vaccines through serum immunoglobulins
Authors: Heatherton Kara , Carey Charleve , Hawkins William , Lacey Stephanie
Published: January 2023

Introduction

The novel SARS-CoV-2 (COVID-19) vaccines generate antibodies to the viral spike protein. The mRNA vaccines Moderna Inc. and Pfizer-BioNTech and adenovirus vaccine Johnson & Johnson (J&J) work through generation of humoral immune responses, particularly immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies. We assessed IgG and IgM response in vaccinated patients longitudinally.

Methods

Blood was drawn from 106 subjects over the course of five time points ranging from pre-to post-vaccine inoculation of Moderna, Pfizer, or J&J. Blood was collected into serum separating tubes (SST). SSTs were spun at 1200rcf for 15 minutes; the serum was isolated and used for testing. Levels of IgG and IgM were measured in geometric mean titers, then analyzed through multiple t-tests across vaccines and demographics.

Results

Moderna vaccine recipients had a significantly higher IgG titer following vaccine inoculation (p < 0.0005 for Time Point 1, 2, 3, 4). No significant difference was observed across various demographics (age/gender) within vaccine types (p > 0.05). There was a steady decline in IgG and IgM levels after full inoculations, which were trending towards baseline levels. J&J recipients had little change in IgG and IgM after receiving the vaccine; however, significantly lower IgG than the Moderna (p = 0.0006) and Pfizer (p =0.0071).

Conclusion

With the use of IgG and IgM as immune response indicator, our data shows Moderna outperforming Pfizer at all Time Points following baseline testing, which suggests greater, longer-lasting immunity against COVID-19. With a low sample size for J&J (n = 5), it is unclear what implications the poor IgG response observed in this study indicates about the adenovirus vaccine.
SARS-CoV-2 Spike395–404 is identified as a murine CD8+ T cell epitope in the receptor-binding domain of the SARS-CoV-2 spike protein
Authors: Yang Jihyun , Kim Eunjin , Poo Haryoung
Published: January 2023

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen of the COVID-19 pandemic. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is considered an important antigen of SARS-CoV-2 vaccines, while the cytotoxic T lymphocyte (CTL) activity is critical for evaluating cell-mediated immune response for vaccine efficacy. Human angiotensin-converting enzyme 2 (hACE2)-expressing transgenic mice with a C57BL/6 genetic background have widely used as an animal model of SARS-CoV-2 study. However, the CTL epitope of SARS-CoV-2 RBD has not yet been identified in C57BL/6 mice and quantifying the CTL activity has used RBD peptide pools with limitations such as expensive and time-consuming synthesis of peptides. In this study, we identified a murine CD8+ T cell epitope of the SARS-CoV-2 spike RBD using C57BL/6 wild-type and hACE2-expressing transgenic (K18-hACE2) mice. Mice were immunized with the adjuvanted SARS-CoV-2 RBD recombinant protein and then the CTL activities were evaluated by determining numbers of the IFN-γ-producing T cells in the splenocytes of the immunized mice upon exposure to overlapping peptide pools or individual peptides via an enzyme-linked immunospot assay and flow cytometry. We found SARS-CoV-2 S395–404 (VYADSFVIRG) as a major histocompatibility complex class I-restricted epitope for the RBD-specific CTL activity in C57BL/6 mice.

Supported by a grant from Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM9942112).
Differential T cell immunity to SARS-CoV-2 in mRNA-1273 and BNT162b2 vaccinated individuals
Authors: Katsis Katelin , Leick Mark B , Larson Rebecca C , Berger Trisha R , Elder Eva L , Maus Marcela V , Gallagher Kathleen M E
Published: January 2023

T-cell immunity to SARS-CoV-2 vaccination with either BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) mRNA vaccines was quantified through a validated ELISpot assay at various time points. The magnitude and duration of response was compared between these two vaccine cohorts alongside the anti-spike IgG response.

Freshly isolated peripheral blood mononuclear cells were incubated with overlapping peptide pools covering the SARS-CoV-2 spike protein in an IFNg ELISpot assay. Donors T cell responses to spike protein were assessed where possible at pre-vaccination, post second vaccination, and at long-term follow up. Anti-Spike IgG was quantified by serum ELISA.

Following the 2 dose regimens, all donors developed anti-spike T-cell responses. The median decline in T-cell response between the 2nd vaccine dose and long-term follow-up was 35.1%. At long-term follow-up, T-cell responses in BNT162b2 vaccinated individuals were lower than mRNA-1273. Median T-cell responses were 38.5 SFU/2.5×105 (BNT162b2) and 86 SFU/2.5×105 (mRNA-1273). Anti-spike IgG decreased by a median of 89.3% at long-term follow-up. Individuals receiving BNT162b2 had lower serum antibody levels long-term compared to mRNA-1273. The median antibody responses were 205.3 BAU/mL (BNT162b2) and 335 BAU/mL (mRNA-1273).

Both humoral and cellular immunity to SARS-CoV-2 is decreased long-term post-vaccination with either mRNA vaccine, and both are significantly lower in BNT162b2-vaccinated compared to mRNA-1273-vaccinated individuals, with a greater magnitude of difference in the T cell response between vaccines compared to antibody levels. As breakthrough infections continue to rise, understanding the dynamics of these vaccine responses is crucial.
MHC Class I–Dependent Shaping of the NK Cell Ly49 Receptor Repertoire Takes Place Early during Maturation in the Bone Marrow
Authors: Wickström Stina L , Wagner Arnika K , Fuchs Sina , Elemans Marjet , Kritikou Joanna , Mehr Ramit , Kärre Klas , Johansson Maria H , Brauner Hanna
Published: January 2023

MHC class I (MHC I) expression in the host influences NK cells in a process termed education. The result of this education is reflected in the responsiveness of NK cells at the level of individual cells as well as in the repertoire of inhibitory MHC I–specific receptors at the NK cell system level. The presence of MHC I molecules in the host environment gives rise to a skewed receptor repertoire in spleen NK cells where subsets expressing few (one or two) inhibitory receptors are expanded whereas subsets with many (three or more) receptors are contracted. It is not known whether this MHC I–dependent skewing is imposed during development or after maturation of NK cells. In this study, we tested the hypothesis that the NK cell receptor repertoire is shaped already early during NK cell development in the bone marrow. We used mice with a repertoire imposed by a single MHC I allele, as well as a C57BL/6 mutant strain with exaggerated repertoire skewing, to investigate Ly49 receptor repertoires at different stages of NK cell differentiation. Our results show that NK cell inhibitory receptor repertoire skewing can indeed be observed in the bone marrow, even during the earliest developmental steps where Ly49 receptors are expressed. This may partly be accounted for by selective proliferation of certain NK cell subsets, but other mechanisms must also be involved. We propose a model for how repertoire skewing is established during a developmental phase in the bone marrow, based on sequential receptor expression as well as selective proliferation.
CD5 Suppresses IL-15–Induced Proliferation of Human Memory CD8+ T Cells by Inhibiting mTOR Pathways
Authors: Choi Young Joon , Lee Hoyoung , Kim Jong Hoon , Kim So-Young , Koh June-Young , Sa Moa , Park Su-Hyung , Shin Eui-Cheol
Published: January 2023

IL-15 induces the proliferation of memory CD8+ T cells as well as NK cells. The expression of CD5 inversely correlates with the IL-15 responsiveness of human memory CD8+ T cells. However, whether CD5 directly regulates IL-15–induced proliferation of human memory CD8+ T cells is unknown. In the current study, we demonstrate that human memory CD8+ T cells in advanced stages of differentiation respond to IL-15 better than human memory CD8+ T cells in stages of less differentiation. We also found that the expression level of CD5 is the best correlate for IL-15 hyporesponsiveness among human memory CD8+ T cells. Importantly, we found that IL-15–induced proliferation of human memory CD8+ T cells is significantly enhanced by blocking CD5 with Abs or knocking down CD5 expression using small interfering RNA, indicating that CD5 directly suppresses the IL-15–induced proliferation of human memory CD8+ T cells. We also found that CD5 inhibits activation of the mTOR pathway, which is required for IL-15–induced proliferation of human memory CD8+ T cells. Taken together, the results indicate that CD5 is not just a correlative marker for IL-15 hyporesponsiveness, but it also directly suppresses IL-15–induced proliferation of human memory CD8+ T cells by inhibiting mTOR pathways.
Cutting Edge: Systemic Autoimmunity in Murine STAT3 Gain-of-Function Syndrome Is Characterized by Effector T Cell Expansion in the Absence of Overt Regulatory T Cell Dysfunction
Authors: Woods Jonathan , Pemberton Sarah E , Largent Andrea D , Chiang Kristy , Liggitt Denny , Oukka Mohamed , Rawlings David J , Jackson Shaun W
Published: January 2023

Germline gain-of-function mutations in the transcriptional factor STAT3 promote early-onset multisystemic autoimmunity. To investigate how increased STAT3 promotes systemic inflammation, we generated a transgenic knock-in strain expressing a pathogenic human mutation STAT3K392R within the endogenous murine locus. As predicted, STAT3K392R mice develop progressive lymphoid hyperplasia and systemic inflammation, mirroring the human disease. However, whereas the prevailing model holds that increased STAT3 activity drives human autoimmunity by dysregulating the balance between regulatory T cells and Th17 cell differentiation, we observed increased Th17 cells in the absence of major defects in regulatory T cell differentiation or function. In addition, STAT3K392R animals exhibited a prominent accumulation of IFN-γ–producing CD4+ and CD8+ T cells. Together, these data provide new insights into this complex human genetic syndrome and highlight the diverse cellular mechanisms by which dysregulated STAT3 activity promotes breaks in immune tolerance.
Systemic Activation of Neutrophils by Immune Complexes Is Critical to IgA Vasculitis
Authors: Mayer-Hain Sarah , Gebhardt Kathleen , Neufeld Matthias , Ehrchen Jan M , Molyneux Karen , Barratt Jonathan , Nattkemper Eva , Gerloff Dennis , Roth Johannes , Vogl Thomas , Pappelbaum Karin I , Sunderkötter Cord
Published: January 2023

In IgA vasculitis (IgAV) perivascular deposition of IgA1 immune complexes (IgA-ICs) is traditionally considered the fundamental trigger for polymorphonuclear neutrophil (PMN)–mediated damage. We propose that IgA-IC deposition, although mandatory, is not sufficient alone for IgAV. Serum IgA-IC levels and IgA-IC binding to PMNs were quantified in IgAV patients and controls. Activation of PMNs was evaluated by neutrophil extracellular trap (NET) release, adherence, and cytotoxicity assays and in a flow system to mirror conditions at postcapillary venules. In vitro results were related to findings in biopsies and a mouse vasculitis model. During acute IgAV flares we observed elevated serum levels of IgA-ICs and increased IgA-IC binding to circulating PMNs. This IgA-IC binding primed PMNs with consequent lowering of the threshold for NETosis, demonstrated by significantly higher release of NETs from PMNs activated in vitro and PMNs from IgAV patients with flares compared with surface IgA-negative PMNs after flares. Blocking of FcαRI abolished these effects, and complement was not essential. In the flow system, marked NETosis only occurred after PMNs had adhered to activated endothelial cells. IgA-IC binding enhanced this PMN tethering and consequent NET-mediated endothelial cell injury. Reflecting these in vitro findings, we visualized NETs in close proximity to endothelial cells and IgA-coated PMNs in tissue sections of IgAV patients. Inhibition of NET formation and knockout of myeloperoxidase in a murine model of IC vasculitis significantly reduced vessel damage in vivo. Binding of IgA-ICs during active IgAV primes PMNs and promotes vessel injury through increased adhesion of PMNs to the endothelium and enhanced NETosis.
Palmitate Potentiates Lipopolysaccharide-Induced IL-6 Production via Coordinated Acetylation of H3K9/H3K18, p300, and RNA Polymerase II
Authors: Kochumon Shihab , Jacob Texy , Koshy Merin , Al-Rashed Fatema , Sindhu Sardar , Al-Ozairi Ebaa , Al-Mulla Fahd , Rosen Evan D , Ahmad Rasheed
Published: January 2023

IL-6 is elevated in obese individuals and participates in the metabolic dysfunction associated with that condition. However, the mechanisms that promote IL-6 expression in obesity are incompletely understood. Because elevated levels of palmitate and LPS have been reported in obesity, we investigated whether these agents interact to potentiate IL-6 production. In this study, we report that LPS induces higher levels of IL-6 in human monocytes in the presence of palmitate. Notably, the priming effect of palmitate is associated with enhanced p300 binding and transcription factor recruitment to Il6 promoter regions. Gene silencing of p300 blocks this action of palmitate. RNA polymerase II recruitment was also enhanced at the Il6 promoter in palmitate/LPS-exposed cells. Acetylation levels of H3K9 and H3K18 were increased in monocytes treated with palmitate. Moreover, LPS stimulation of palmitate-treated cells led to increased levels of the transcriptionally permissive acetylation marks H3K9/H3K18 in the Il6 promoter compared with LPS alone. The effect of palmitate on LPS-induced IL-6 production was suppressed by the inhibition of histone acetyltransferases. Conversely, histone deacetylase inhibitors trichostatin A or sodium butyrate can substitute for palmitate in IL-6 production. Esterification of palmitate with CoA was involved, whereas β-oxidation and ceramide biosynthesis were not required, for the induction of IL-6 and H3K9/H3K18 acetylation. Monocytes of obese individuals showed significantly higher H3K9/H3K18 acetylation and Il6 expression. Overall, our findings support a model in which increased levels of palmitate in obesity create a setting for LPS to potentiate IL-6 production via chromatin remodeling, enabling palmitate to contribute to metabolic inflammation.
Identification of a Small Molecule with Strong Anti-Inflammatory Activity in Experimental Autoimmune Encephalomyelitis and Sepsis through Blocking Gasdermin D Activation
Authors: Cao Runjing , Li Zihao , Wu Chuyu , Ji Senlin , Li Yahui , Cao Xiang , Dong Xiaohong , Jiang Meiling , Pang Tao , Wang Chenhui , Li Jingwei , Xu Yun , Zhang Cun-Jin
Published: January 2023

Pyroptosis is a key inflammatory form of cell death participating in the progression of many inflammatory diseases, such as experimental autoimmune encephalomyelitis (EAE) and sepsis. Identification of small molecules to inhibit pyroptosis is emerging as an attractive strategy. In this study, we performed a screening based on in silico docking of compounds on the reported Gasdermin D (GSDMD) three-dimensional structure and found C202-2729 demonstrated strong anti-inflammatory effects in both endotoxin shock and EAE mouse models. Oral administration of C202-2729 was capable of attenuating EAE disease severity significantly and has the comparable effects to teriflunomide, the first-line clinical drug of multiple sclerosis. We found C202-2729 remarkably suppressed macrophage and T cell–associated immune inflammation. Mechanistically, C202-2729 neither impact GSDMD cleavage nor the upstream inflammasome activation in mouse immortalized bone marrow–derived macrophages. However, C202-2729 exposure significantly repressed the IL-1β secretion and cell pyroptosis. We found C202-2729 directly bonds to the N terminus of GSDMD and blocks the migration of the N-terminal GSDMD fragment to cell membrane, restraining the pore-forming and mature IL-1β release. Collectively, our findings provide a new molecule with the potential for translational application in GSDMD-associated inflammatory diseases.
The Foot-and-Mouth Disease Virus Lb Protease Cleaves Intracellular Transcription Factors STAT1 and STAT2 to Antagonize IFN-β–Induced Signaling
Authors: Ma XuSheng , Luo ZhiKuan , Song Rui , Nian XiaoFeng , Choudhury Sk Mohiuddin , Ru Yi , Yang Fan , Zhang YuXia , Zeng ZongBo , Cao WeiJun , Pei JingJing , Liu XiangTao , Zheng HaiXue
Published: January 2023

Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, one of the most highly infectious animal viruses throughout the world. The JAK-STAT signaling pathway is a highly conserved pathway for IFN-β–induced antiviral gene expression. Previous studies have shown that FMDV can strongly suppress the innate immune response. Moreover, although STAT1 and STAT2 (STAT1/2) have been well established in JAK-STAT signaling–induced antiviral gene expression, whether FMDV proteins inhibit IFN-β–induced JAK-STAT signaling remains poorly understood. In this study, we described the Lb leader protease (Lbpro) of FMDV as a candidate for inhibiting IFN-β–induced signaling transduction via directly interacting with STAT1/2. We further showed that Lbpro colocalized with STAT1/2 to inhibit their nuclear translocation. Importantly, Lbpro cleaved STAT1/2 to inhibit IFN-β–induced signal transduction, whereas the catalytically inactive mutant of LC51A (Lbpro with cysteine substituted with alanine at amino acid residue 51) had no effect on the stability of STAT1/2 proteins. The cleavage of the STAT1/2 proteins was also determined during FMDV infection in vitro. Lbpro could cleave the residues between 252 and 502 aa for STAT1 and the site spanning residues 140 − 150 aa (QQHEIESRIL) for STAT2. The in vivo results showed that Lbpro can cleave STAT1/2 in pigs. Overall, our findings suggest that FMDV Lbpro–mediated targeting of STAT1/2 may reveal a novel mechanism for viral immune evasion.
A Vitamin D–RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN–IRF7 Signaling
Authors: Ratra Yashika , Kumar Naveen , Saha Manti K , Bharadwaj Chandrima , Chongtham Chen , Bais Sachendra S , Medigeshi Guruprasad , Arimbasseri Gopalakrishnan A , Basak Soumen
Published: January 2023

Besides its functions in the skeletomuscular system, vitamin D is known to alleviate viral-inflicted pathologies. However, the mechanism underlying protective vitamin D function remains unclear. We examined the role of vitamin D in controlling cellular infections by Chandipura virus, an RNA virus implicated in human epidemics. How immune signaling pathways, including those regulating NF-κB and IFN regulatory factors (IRFs), are activated in virus-infected cells has been well studied. Our investigation involving human- and mouse-derived cells revealed that vitamin D instructs the homeostatic state of these antiviral pathways, leading to cellular resilience to subsequent viral infections. In particular, vitamin D provoked autoregulatory type 1 IFN–IRF7 signaling even in the absence of virus infection by downmodulating the expression of the IFN-inhibitory NF-κB subunit RelB. Indeed, RelB deficiency rendered vitamin D treatment redundant, whereas IRF7 depletion abrogated antiviral vitamin D action. In sum, immune signaling homeostasis appears to connect micronutrients to antiviral immunity at the cellular level. The proposed link may have a bearing on shaping public health policy during an outbreak.