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Introduction Patients with hematological malignancies, including multiple myeloma (MM), experience suboptimal responses to SARS-CoV-2 vaccination. Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM) are precursors to MM and exhibit altered immune cell composition and function. The SARS-CoV-2 pandemic and the subsequent population-wide vaccination represent an opportunity to study the real-life immune response to a common antigen. Here, we present updated results from the IMPACT study, a study we launched in November 2020 to characterize the effect of plasma cell premalignancy on response to SARS-CoV2 vaccination (vx). Methods We performed: (i) ELISA for SARS-CoV-2-specific antibodies on 1,887 peripheral blood (PB) samples (237 healthy donors (HD), and 550 MGUS, 947 SMM, and 153 MM patients) drawn preand post-vx;(ii) single-cell RNA, T cell receptor (TCR), and B cell receptor (BCR) sequencing (10x Genomics) on 224 PB samples (26 HD, and 20 MGUS, 48 SMM, and 24 MM patients) drawn preand post-vx;(iii) plasma cytokine profiling (Olink) on 106 PB samples (32 HD, and 38 MGUS and 36 SMM patients) drawn pre- and post-vx;and (iv) bulk TCR sequencing (Adaptive Biotechnologies) on 8 PB samples from 4 patients (2 MGUS, 2 SMM) drawn pre- and post-vx. Results Patients with MGUS and SMM achieved comparable antibody titers to HD two months post-vx. However, patient titers waned significantly faster, and 4 months post-vx we observed significantly lower titers in both MGUS (Wilcoxon rank-sum, p=0.030) and SMM (p=0.010). These results indicate impaired humoral immune response in patients with MGUS and SMM.At baseline, the TCR repertoire was significantly less diverse in patients with SMM compared to HD (Wilcoxon rank-sum, p=0.039), while no significant difference was observed in the BCR repertoire (p=0.095). Interestingly, a significant increase in TCR repertoire diversity was observed post-vx in patients with SMM (paired t-test, p=0.014), indicating rare T cell clone recruitment in response to vaccination. In both HD and patients, recruited clones showed upregulation of genes associated with CD4+ naive and memory T cells, suggesting preservation of the T cell response in SMM, which was confirmed by bulk TCR-sequencing in 4 patients.Lastly, by cytokine profiling, we observed a defect in IL-1beta and IL-18 induction post-vx in patients with SMM compared to HD (Wilcoxon rank-sum, p=0.047 and p=0.015, respectively), two key monocyte-derived mediators of acute inflammation, suggesting an altered innate immune response as well. Conclusion Taken together, our findings highlight that despite the absence of clinical manifestations, plasma cell premalignancy is associated with defects in both innate and adaptive immune responses. Therefore, patients with plasma cell premalignancy may require adjusted vaccination strategies for optimal immunization.
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Background & Aim: The new UCOE models we have recently developed, tested on many cell groups (including mouse ES and human iPS cells) and human mAb recombinant production studies as well, shows a powerful resistance to DNA methylation- mediated silencing and provides a higher and stable transfection profile. By the urgent need of vaccine development for COVID-19 during the pandemic, in this study we aimed to produce a potential recombinant vaccine by using the new generation UCOEs models of our own design. Methods, Results & Conclusion(s): Existing new-generation UCOE models and standard plasmid vectors to be used as control group were provided. Then, the sequences related to the PCR method were amplified for sufficient stock generation and cloning experiments. Verification in the plasmid vector was carried out in gel electrophoresis. Transfection of 293T cells was performed with clone plasmids carrying antigen genes and plasmids carrying genetic information of lentivirus units for the production of lentiviral vectors. Afterwards, 293T cells produced lentiviral vectors carrying antigen genes. Harvesting of these vectors was carried out during 48th and 72nd hours. Afterwards, CHO cells were transduced with appropriate quantity of lentiviral vectors. Isolation and purification of targeted proteins from the relevant medium were performed by HPLC and Q-TOF methods. A part of the spike and nucleocapsid gene sequences of COVID-19 were firstly cloned into our UCOE models. These UCOEs plasmids were then transferred into 293T cells along with plasmids carrying the genes that will form the lentivirus vectors (LVs). After harvesting and calculation of LV vector titers, the cloned vectors were then transfected into the CHO cells which the targeted recombinant production of the antigen proteins will be carried out. Antigenic structures were then isolated from the culture medium of CHO cells in following days for confirmation. Using HPLC and qTOF mass spectrometer methods, these structures in the medium were confirmed to be the units of spike and nucleocapsid proteins of the COVID-19 virus. In order to produce large amount of the recombinant antigens, the culture was then carried out with bioreactors in liters. At the final stage, these recombinantly produced antigen proteins were tested on rats to measure their immunogenic responses, and the study recently been completed successfully as a potential recombinant vaccine against COVID-19.Copyright © 2023 International Society for Cell & Gene Therapy
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Background: Hybrid immunity is more protective than vaccination or prior infection alone. To understand the formation of hybrid immunity, we studied how SARS-CoV-2 mRNA vaccines interact with T cell memory by tracking spike (S) specific T cells in cohorts of hospitalized (n = 19) or non-hospitalized (n = 34) COVID-19 convalescents. We hypothesized that S-reactive CD4 and CD8 T cells would increase in response to serial vaccine doses and reflect prior immune exposure at the clonal level. Method(s): After vaccination, we stimulated PBMCs from 12 participants (8M/4F) with peptides spanning S. Activated cells (CD69+CD137+) were sorted and CD4/CD8 phenotype linked with paired TRB-TRA sequences at single cell resolution. S-reactive TRB sequences were mapped within 4-6 serial blood and post-booster nasal TRB repertoires to evaluate S-reactive CD4 and CD8 T cell clonotypic kinetics spanning convalescence to boost. PBMCs from 53 participants were sequenced with the ImmunoSEQ assay to evaluate S-reactive TRB breadth using a database of S-assigned TRB sequences (Adaptive Biotechnologies), comparing S-reactive TRB diagnostic breadth by hospitalization status (Wilcoxon test). Result(s): SARS-CoV-2 mRNA vaccination provoked strong T cell clonal expansion in most participants. At 8-12 months after infection, each primary mRNA dose increased the abundance and diversity of S-specific T cells. Clonal and integrated expansions were larger in CD8 than in CD4 T cells. At the convalescent time point, we observed greater diagnostic S-reactive CD4 T cell breadth in hospitalized vs. non-hospitalized patients (p< 0.01). CD4 T cell S breadth was again higher in previously hospitalized persons after the 2nd primary (p=0.02) and booster (p< 0.01) doses, suggesting that diverse CD4 T cell memory after severe infection leads to increased repertoire diversity after vaccination. S-specific T cells with identical TCRs were detectable in blood and the nasal mucosa, with specificity confirmed using a TRA/TRB transgenic T cell with the matching receptor. Conclusion(s): Although both S-specific CD8 and CD4 T cell memory are established by prior infection, S-specific CD8 T cells predominated in blood after primary vaccination, with some clonotypes showing up to 1000-fold expansion across 1-2 mRNA doses. Vaccine-reactive CD8 clonotypes were present at the barrier nasal site after booster mRNA dosing. Severe disease imprinted a highly diverse S-reactive CD4 repertoire persisting through vaccination.
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Background: T cells play a critical role in the adaptive immune response to SARS-CoV-2 in both infection and vaccination. Identifying T cell epitopes and understanding how T cells recognize these epitopes can help inform future vaccine design and provide insight into T cell recognition of newly emerging variants. Here, we identified SARS-CoV-2 specific T cell epitopes, analyzed epitope-specific T cell repertoires, and characterized the potency and cross-reactivity of T cell clones across different common human coronaviruses (HCoVs). Method(s): SARS-CoV-2-specific T cell epitopes were determined by IFNgamma ELISpot using PBMC from convalescent individuals with mild/moderate disease (n=25 for Spike (S), Nucleocapsid (N) and Membrane (M)), and in vaccinated individuals (n=27 for S). Epitope-specific T cells were isolated based on activation markers following a 6-hour peptide stimulation, and scRNAseq was performed for TCR repertoire analysis. T cell lines were generated by expressing recombinant TCRs in Jurkat cells and activation was measured by CD69 upregulation. Result(s): We identified multiple immunodominant T cell epitopes across S, N and M proteins in convalescent individuals. In vaccinated individuals, we detected many of the same dominant S-specific epitopes at similar frequencies as compared to convalescent individuals. T cell responses to peptide S205 (amino acids 817-831) were observed in 56% and 59% of individuals following infection and vaccination, respectively, while 20% and 19% of individuals responded to S302 (a.a. 1205-1219) following infection and vaccination, respectively. For S205, a CD4+ T cell response, we confirmed 8 unique TCRs and determined the minimal epitope to be a 9mer (IEDLLFNKV). While TCR genes TRAV8-6*01 and TRBV30*01 were commonly utilized across the TCRs, we did identify TCRs with unique immunogenetic properties with different potencies of cross-reactivity to other HCoVs. For S302, a CD8+ T cell response, we identified two unique TCRs with different immunogenetic properties that recognized the same 9mer (YIKWPWYIW) and cross-reacted with different HCoV peptides (Figure 1). Conclusion(s): These data identify immunodominant T cell epitopes following SARS-CoV-2 infection and vaccination and provide a detailed analysis of epitope-specific TCR repertoires. The prospect of developing a vaccine that broadly protects against multiple human coronaviruses is bolstered by the identification of conserved immunodominant SARS-CoV-2 T cell epitopes that cross react with multiple other HCoVs.
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It has been proven that mRNA vaccines are highly effective against the COVID-19 outbreak, and low prevalence of side effects has been shown. However, there are still many gaps in our understanding of the biology and biosafety of nucleic acids as components of lipid nanoparticles (LNPs) most often used as a system for inctracellular delivery of mRNA-based vaccines. It is known that LNPs cause severe injection site inflammation, have broad biodistribution profiles, and are found in multiple tissues of the body, including the brain, after administration. The role of new medications with such pharmacokinetics in inflammation developing in inaccessible organs is poorly understood. The study was aimed to assess the effects of various doses of mRNA-LNP expressing the reporter protein (0, 5, 10, and 20 microg of mRNA encoding the firefly luciferase) on the expression of neuroinflammation markers (Tnfalpha, Il1beta, Gfap, Aif1) in the prefrontal cortex and hypothalamus of laboratory animals 4, 8, and 30 h after the intramuscular injection of LNP nanoemulsion. It was shown that mRNA-LNP vaccines in a dose of 10-20 microg of mRNA could enhance Aif1 expression in the hypothalamus 8 h after vaccination, however, no such differences were observed after 30 h. It was found that the Gfap, l11beta, Tnfalpha expression levels in the hypothalamus observed at different times in the experimental groups were different. According to the results, mRNA-LNPs administered by the parenteral route can stimulate temporary activation of microglia in certain time intervals in the dose-dependent and site specific manner.Copyright © 2022 Pirogov Russian National Research Medical University. All rights reserved.
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Introduction: Some MS DMTs, such as anti-CD20 antibodies and sphingosine-1-phosphate (S1P) receptor modulators, decrease post-infection and post-vaccination SARS-Cov-2 humoral responses. However, humoral immunity is only one component of the adaptive response, and T cell responses, which may be preserved in anti-CD20 treated patients, play an important role. Objective(s): To characterize SARS-Cov-2 spike specific memory T-cell receptor repertoires in patients with MS and related conditions post-vaccination with mRNA vaccines. Aim(s): To characterize SARS-Cov-2 vaccine-mediated responses for people using MS DMTs. Method(s): Patients without prior COVID-19 infection provided a whole blood sample >3 weeks and <6 months after vaccination with two doses of Pfizer-BioNTech or Moderna mRNA vaccines. Sequencing of the complementary determining region within T-cell receptors (TCRs) was performed. Antigen recognition activates unique TCRs and results in expansion of antigen-specific T-cell clones. Sample TCR sequences were cross-matched with sequences known to react to SARS-CoV-2 using "Multiplex Identification of T-cell Receptor Antigen Specificity (MIRA)", allowing for characterization of SARS-CoV-2-spike-specific TCR frequency (clonal depth) and diversity (clonal breadth). Humoral responses were compared. Result(s): 39 patients were recruited: age 25-77;27 female;37 with MS, 2 with NMO, and 1 with another neuroimmune condition. DMTs included anti-CD20 (N=13), natalizumab (N=9), fumarates (N=8), S1P receptor modulators (N=3), and controls (2 glatiramer acetate, 4 no DMT). Mean time interval between 2nd vaccination dose and TCR testing was 13.3+6.0 weeks. Humoral responses (Roche) were absent in all anti-CD20 and S1P treated patients but preserved in all others. SARS-CoV-2-spikespecific clonal depth and breadth did not differ across all treatment classes except S1P modulators. Despite lack of antibody production, patients treated with anti-CD20 therapies demonstrated comparable TCR depth and breadth to all other groups in univariable assessment. No spike-specific TCRs were found in patients treated with S1P modulators. TCR breadth and depth did not vary with time since vaccination even up to 24 weeks following vaccination. Conclusion(s): TCR repertoires were preserved except for in those treated with S1P receptor modulators.Humoral responses were diminished with both anti-CD20 and S1P DMTs. These findings may help guide counseling of patients with regards to DMT choice.
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Introduction: Infection with the SARS-CoV-2 coronavirus can lead to a wide range of acute and also chronic disease manifestations. The rapidly developed vaccinations are highly effective in preventing severe disease courses and have been proven safe. Both natural infection and, to a much lower extent, the mRNAbased vaccinations can be accompanied by transient autoimmune phenomena or onset of autoimmune diseases. Objective(s): We report here two cases of multiple sclerosis (MS) with clinical and new radiological signs beginning in close temporal relation to spike (S) protein mRNA-based vaccinations. Aim(s): To establish that the onset of MS in these two cases is very likely caused by CD4+ T cell clones that cross-recognize SARSCoV- 2 S protein-derived peptides and peptides derived from myelin proteins, which have previously been implicated in MS. Method(s): Spike specific CD4+ T cells from peripheral blood and CD4+ T cells from CSF sample were isolated and expanded for autoantigen screening test. A list of well-known MS-related autoantigens including immunodominant peptides and isoforms from MBP, MOG, PLP, RASGRP2, TSTA3 peptides were included to assess T cell reactivity. CD4+ CFSElow fraction were sorted after stimulate with positive autoantigen pools or SARSCov- 2 Spike protein, followed by expansion and testing with autoantigen peptides and Spike protein. Supernatant from cell culture were further analyzed for IFN-gamma secretion. Result(s): Self-reactive T cells were detected from Spike specific T cell population in both patients. CD4+ T from CSF also showed reactivity to MBP, MOG, PLP peptide pools. Finally, we found proinflammatory T cell clones that recognize both Spike protein and immunodominant MBP peptides and MOG peptides, which have previously been implicated in MS. Conclusion(s): Detailed studies of both peripheral blood- and CSFderived CD4+ T cells show that the onset of MS in these two cases is very likely caused by CD4+ T cell clones that cross-recognize SARS-CoV-2 S protein-derived peptides and peptides derived from myelin proteins, which have previously been implicated in MS.
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Introduction: Kidney involvement is common in multiple myeloma (MM) and is associated with increased mortality. Light chain cast nephropathy, monoclonal immunoglobulin deposition disease and light chain amyloidosis are the most frequent presentations. Very few cases are reported of MM presenting as crescentic glomerulonephritis (CGN). Here, we present a case of a female with rapidly progressive loss of renal function and proteinuria who was found to have MM and CGN. Case Description: A 45-year old woman with h/o hypertension, diabetes mellitus, GERD, iron deficiency anemia, nephrolithiasis and COVID infection six months presented with worsening renal function. Her baseline creatinine three months prior was 0.88 mg/dL eGFR >60 and had recently increased to 2.81 mg/dL eGFR of 22. Laboratory investigations revealed a hemoglobin 8.3 g/dL, platelet count 404 x 10x9/L. Serum calcium and uric acid levels were within normal limits. Urinalysis showed proteinuria without hematuria with a urine protein/creatinine ratio 1.9 g/g, urine albumin/creatinine ratio 506 mg/g and urine albumin/protein ratio of 0.27. Complement levels, ANCA, Anti-GBM, ANA, RF and hepatitis serology were all unrevealing. Serum Kappa and Lambda free light chains were elevated with ratio being of 0.09. Bone marrow was done which demonstrated approximately 10% lambda restricted plasma cell clones. In view of progressive renal dysfunction she was admitted with the suspicious of light chain nephropathy for consideration of plasma exchange therapy. Kidney biopsy was obtained which showed findings consistent with crescentic GN, with weak linear IgG staining along glomerular basement membranes without evidence of cast nephropathy, monoclonal immunoglobulin deposition or amyloidosis. The patient was started on Cyclophosphamide-Bortezomib-Dexamethasone regimen and since then her kidney function has remained stable. Discussion(s): Rare kidney findings in patients with MM include membranoproliferative GN, cryoglobulinemia, immunotactoid and fibrillary glomerulopathy. MM associated Crescentic GN is extremely rare. Its etiology and pathophysiology is unclear but it seems that treatment of MM may temporarily halt its progression as in this case.
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Background: Mantle cell lymphoma (MCL) is a B-cell tumor which often relapses. BCR inhibitors (Ibrutinib, Acalabrutinib) and antiapoptotic BCL2-family members blockers BH3-mimetics (Venetoclax, ABT-199) are effective drugs to fight MCL. However, the disease remains incurable, due to therapy resistance, even to the promising Venetoclax and Ibrutinib combination. Therefore, there is a profound need to explore novel useful therapeutic targets. CK2 is a S/T kinase overexpressed in several solid and blood tumors. We demonstrated that CK2, operating through a 'non-oncogene addiction' mechanism promotes tumor cell survival, and counteracts apoptosis, by activating pro-survival signaling cascades, such as NF-κ B, STAT3 and AKT. CK2 could regulate also BCL2 family members. The CK2 chemical inhibitor CX-4945 (Silmitasertib, Sil) is already under scrutiny in clinical trials in relapsed multiple myeloma, solid tumors and COVID-19. Aims: In this work, we tested the effect of CK2 chemical inhibition or knock down on Venetoclax (Ven)-induced cytotoxicity in MCL pre-clinical models to effectively reduce MCL cell growth and clonal expansion. Methods: CK2 expression and BCR/BCL2 related signaling components were analyzed in MCL cells and control cells by Western blotting. CK2 and BCL2 inhibition was obtained with Sil and Ven, respectively and with CK2 gene silencing through the generation of anti-CK2 shRNA IPTG-inducible MCL cell clones. Survival, apoptosis, mitochondrial membrane depolarization and proliferation were investigated by FACS analysis of AnnexinV/PI and JC-10 staining. The synergic action of Ven and Sil was analyzed by the Chou-Talalay combination index (CI) method. CK2 knock down in vivo was obtained in xenograft NOD-SCID mouse models Results: CK2 inactivation (with Sil or CK2 silencing) determined a reduction in the activating phosphorylation of S529 p65/RelA and S473 and S129 AKT, important survival cascades for MCL. Sil or CK2 silencing caused BCL2 and related MCL1 protein reduction, causing cell death. Importantly, we confirmed these results also in an in vivo xenograft mouse model of CK2 knockdown in MCL. Sil +Ven combination increased MCL cell apoptosis, as judged by the augmented frequency of Annexin V positive cells and expression of cleaved PARP protein, and JC-10 mitochondrial membrane depolarization, with respect to the single treatments. Captivatingly, Sil or CK2 gene silencing led to a substantial reduction of the Ven-induced increase of MCL-1, potentially counteracting a deleterious Ven-induced drawback. Analysis of cell cycle distribution confirmed an increased frequency of apoptotic cells in the sub G1 phase in CK2-silenced cells and a modulation of the other phases of the cell cycle. Remarkably, the calculated CI less than 1 suggested a strong synergic cell-killing effect between Sil and Ven, on all the cell lines tested, including those less sensitive or resistant to Ven Summary/Conclusion: We demonstrated that the simultaneous inhibition/knock down of CK2 and BCL2 synergistically cooperates in inducing apoptosis and cell cycle arrest of MCL malignant B-lymphocytes and has the potential of reducing MCL clonal growth, also counterbalancing mechanism of resistance that may arise with Ven. Therefore, CK2 is a rational therapeutic target for the treatment of MCL to be tested in combination with Ibrutinib or Ven.
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Recent clinical observations that some coronavirus infections induced complete remissions in lymphoma patients emphasized again the potential of cancer virotherapy. Infection of cancer cells with oncolytic viruses reshapes the tumor microenvironment by activating anti-viral and anti-tumor immunity. A phase 1 clinical trial using oncolytic adenovirus Delta-24-RGD (DNX-2401) to treat recurrent malignant gliomas demonstrated activation of CD8+ T-cells and significant clinical benefits for a subset of patients. However, both anti-virus and anti-tumor immune responses are contingent on the activation of respective clones of CD8+ T-cells, which compete for clonal expansion. Thus, overexpansion of T-cells against viral antigens reduces the frequency of subdominant clones against tumor antigens. We hypothesized that inducing immune tolerance for viral antigens will decrease anti-viral immunity and in turn derepress anti-tumor immunity, resulting in enhanced efficacy of cancer virotherapy. In this work, we used nanoparticles encapsulating adenoviral antigens E1A, E1B and hexon that distributed to liver resident macrophages (P<0.0001) and induced peripheral immune tolerance. Functional experiments to restimulate immune cells with viral or tumor antigens showed that injection of nanoparticles induced virus-specific immune tolerance and redirected the focus of the immune response towards tumor peptides as measured by interferon-gamma secretion (P<0.0001). Co-culture experiments also showed increased activation of immune cells against fixed tumor cells after nanoparticle treatment (P<0.0001). Reduction of virus-specific T-cells and concurrent expansion of tumor-specific T-cell clones were further confirmed with E1A or OVA tetramers (P<0.05). Flow cytometry analysis suggested increased anti-tumor responses were due to differences in T-cell clones and not due to other immune populations including natural killer cells or myeloid-derived suppressor cells (P=0.3). Importantly, virotherapy in combination with nanoparticle-induced immune tolerance towards viral antigens in tumor-bearing mice increased the overall survival and doubled the percentage of long-term survivors compared to virus treatment alone. Our data should propel the development of a future clinical trial aiming to maximize the potential of anti-tumor immunity during cancer virotherapies.
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Background: Rapid and large-scale deployment of COVID-19 mRNA vaccines highlights the potential utility of developing nucleic acid vaccines (such as RNA and DNA vaccines) against infectious diseases, including HIV-1. However, as compared to SARS-CoV-2, HIV-1 pose some unique challenges-induction of neutralizing antibodies (NAbs) against HIV-1 (frequently a correlate of protection) requires presentation of trimeric and highly conformational epitopes to the immune system, and whether nucleic acid vaccines can enable direct in vivo production of antigens that retain critical antigenic profile has not yet been elucidated. Additionally, it was previously reported that Tier 2 NAbs cannot be induced in mice due to a lack of antibody repertoire, and vaccine studies were suggested to be performed in larger mammals such as rabbits/NHPs, inadvertently slowing down and increasing the costs of preclinical HIV-1 vaccine studies. Methods: In our study, we used the Antigen Conformation Tracing In Vivo by ELISA (ACTIVE) assay developed in house to characterize antigenic profiles of vaccines produced in vivo (from transfected muscle tissues). We analyzed induced cellular responses, using stimulation with overlapping peptides followed by intracellular cytokine staining and IFN-g ELIspot assays. We analyzed induced humoral responses by using both binding ELISA assays and TZM-BL based neutralizing assays, and attempted to map induced NAb epitopes by engineering selectively mutated pseudovirus. We performed antigen-specific B-cell sorting, and used the 10x genomics pipeline to characterize antibody sequences of proliferating B-cell clones. Results: We confirmed that in vivo produced vaccines retained key trimeric conformational epitopes and glycan profiles. Compared to protein vaccination, DNA vaccination uniquely and strongly induced both TFH, CD4+, CD8+ T-cell responses, and Tier 2 NAbs mapped to a previously unreported Env C3/V5 epitope. 5 unique NAbs were isolated, and confirmed to bind to the epitope using a Cryo-EM structure of NAb-MD39 complex at 3.8Å resolution. Conclusion: Our study confirmed that with appropriate vaccine delivery technology, murine models can be appropriately used for HIV-1 vaccine studies aimed at generating NAb responses. In addition, beyond potential functional immunity gains, DNA vaccines permit in vivo folding of structured antigens and provide significant cost and speed advantages for enabling rapid evaluation of new HIV vaccines.
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Background: Recent studies have shown that vaccinated individuals harbor cross-reactive T cell responses that can cross-recognize SARS-CoV-2 and endemic human common cold coronaviruses (HCoVs). However, it is still unknown whether CD4+ T cells from vaccinated individuals recognize peptides from bat coronaviruses that may have the potential of causing future pandemics. In this study, we identified a SARS-CoV-2 spike protein epitope (S815-827) that is conserved in coronaviruses from different genera and subgenera including SARS-CoV, MERS-CoV, multiple bat coronaviruses and a feline coronavirus. We hypothesized that S815-827 is recognized by vaccinated individuals, and that S815-827-reactive T cells can cross-recognize homologues bat coronaviruses. Methods: To evaluate CD4+ T cell responses, we isolated CD8 depleted PBMCs from COVID-19 vaccinated individuals and performed IFN-γ ELISPOT assays. To assess T cell cross-reactivity, S815-827-reactive T cell lines were re-stimulated with homologous coronavirus peptides and cytokine production was assessed with flow cytometry. Additionally, the Vira-FEST assay (which utilizes TCR Vβ CDR3 sequencing) was performed to identify cross-reactive CD4+ T cell clones. Statistical comparisons were done using Mann-Whitney test, Wilcoxon matched-pairs signed rank test or Friedman test with Dunn's multiple comparison as appropriate. Results: Our results show that 16 out of 38 (42%) of vaccinated participants in our study who received the Pfizer-BioNTech (BNT162b2) or Moderna (mRNA-1273) COVID-19 vaccines had robust CD4+ T cell responses to S815-827. All responders also recognized homologous peptides from at least 2 other coronaviruses, and 8 out of 11 responders recognized peptides from at least 6 out of the 9 other coronaviruses tested. To determine T cell cross-reactivity, we re-stimulated S815-827 specific T cell lines with homologous coronavirus peptides. We found that S815-827 specific T cells had a robust increase in IFN-γ+ TNF-α+ expression upon re-stimulation with other peptides. We next used the Vira-FEST assay to confirm cross-reactivity by assessing if the same CD4+ T receptor clonotypes recognize both S815-827 and homologous bat coronavirus peptides. In all 3 participants tested, we identified multiple cross-reactive T cell receptors that recognize both S815-827 and homologous bat coronavirus peptides. Conclusion: Our results suggest that current mRNA vaccines elicit T cell responses that can cross-recognize bat coronaviruses, and thus might induce protection.
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Introduction: Since December 2019, COVID-19 has spread rapidly across the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as patients after allogeneic haematopoietic stem cell transplantation. Immunological protection against COVID-19 is mediated by both shortlived neutralising antibodies and long-lasting virus-reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Methods: Activation of CD4+ T cells from 18 COVID-19 patients was determined by flow cytometry, both ex vivo and after in vitro restimulation with SARS-CoV-2 Spike and Nucleocapsid antigens. Immunodominant, 15-mer peptides were identified using epitope mapping. T cells clones specific for these epitopes were further chararacterised for the sensitivity and polarisation of their cytokine responses after in vitro restimulation, by ELISA and cytometric assay. Next-generation sequencing revealed fulllength, paired T Cell Receptor (TCR) αβ sequences. Results: We identified three patients with strong CD4+ T cells to SARSCoV- 2 antigens. From these patients, 81 T cell clones specific for a selection of 9 immunodominant epitopes (7 Spike and 2 Nucleocapsid epitopes) were generated. Cytokine analysis showed that the sensitivity and polarisation of T cell responses varied depending on the specific epitope. Moreover, TCRαβ sequences revealed an epitope-dependent difference in the level of clonality. Conclusions: We provide detailed information on SARS-CoV-2-specific CD4+ T cells, including their antigen-specificity, the nature of their cytokine responses and the full sequence of their TCRαβ. These cells have the potential to direct an effective immune response in COVID-19 patients. Our results form a crucial first step towards T cell therapy. Efforts are underway to develop transgenic CD4+ T cells that express the SARS-CoV- 2-specific TCRs identified.
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Patients with lymphoproliferative disorders are at high risk for severe COVID-19. For patients with AL amyloidosis, in which there is also critical organ involvement, this risk may be even higher. Vaccination against SARS-CoV-2 is the best strategy to avoid severe COVID-19, but response to vaccines may be compromised in patients with B-cell lymphoproliferative or plasma cell malignancies, as in AL amyloidosis. Although modest in size, the plasma cell clone in AL may cause immunosuppression while anticlonal therapies further compromise immune responses. To evaluate immunization efficacy, we measured the titers of neutralizing antibodies (NAbs) against SARS-CoV-2 after vaccination with BNT162b2 in patients with AL amyloidosis. As a control group we used volunteers, matched (ratio 1:2) for age and gender, who had no autoimmune or active malignant or infectious disease. Serum was separated within 4 hours from blood collection and stored at -80°C until the day of measurement on (A) day 1 (D1;before the first dose of BNT162b2) (B) day 22 (D22;before the 2nd dose) and (C) day 50 (D50;ie 30 days after the 2nd dose). NAbs against SARS-CoV-2 were measured using FDA approved methodology (cPass™ SARS-CoV-2 NAbs Detection Kit;GenScript, Piscataway, NJ, USA). According to the manufacturer of the assay, a titer ≥ 50% is considered a clinically relevant threshold for viral inhibition. The study included 144 patients with AL amyloidosis, of which 120 had NAbs titers assessed on all time points and were included in the final analysis (53% males;median age: 66, IQR: 57-72 years) and 240 matched controls (53% males;median age: 66, IQR: 57-72 years). 66 (55%) AL patients were on active therapy, 17.5% were on daratumumab (DARA)-based therapy, 52 (43%) had discontinued therapy >3 months from the date of the first shot, 19% had prior exposure to DARA and 94 (78%) were in hematologic remission (CR or VGPR). Prior to the 1st dose (D1), NAb titers were similar between patients and controls (median 14.9% (IQR 7.8-23.1%) vs 14% (IQR 6.8-22.9%), p=0.439);6 AL patients had baseline NAbs >50%, of which 5 reported a history of COVID-19 infection. On D22, there was a significant increase of NAbs titers both in controls and AL patients (both p<0.001);however, median NAb titer was 23.6% (IQR 12.4-37.7%) in AL patients vs 47.5% (IQR 32.1-62.7%) in controls (p<0.001) and 20.5% of AL patients vs 46.7% of controls (p<0.001) developed NAb titers ≥50%. On D50, there was further increase in NAbs titers both in controls and AL patients (both p<0.001) and median NAb titer for AL patients was 83.1% (IQR 41.5-94.9%) vs 95.6% (IQR 91.7-97.2%) in controls (p<0.001);71% of AL patients vs 98% of matched controls (p<0.001) developed NAb titers ≥50%. Among AL patients, factors associated with NAb titers on D50 included age (p<0.001), lymphocyte counts (p<0.001), serum albumin (p<0.001) and amount of proteinuria at the time of vaccination (p=0.047), renal involvement (p=0.047), use of steroids (p<0.001), active treatment (p<0.001), treatment-free interval (p=0.001), remission status (CR/VGPR) (p=0.018). There was no significant association with gender (p=0.092), BMI (p=0.198), IgG (0.099), IgA (p=0.789) or IgM levels (p=0.687), liver (p=0.521) or heart involvement (p=0.141). Patients on therapy had lower NAb titers at D50 (median 50.1% (IQR 25.3-84.1%) vs 91.6% (IQR 74.5-96.5%) for those not on treatment, p<0.001), so that 51% had a D50 NAb titer ≥50% vs 87% of those not on therapy. Current DARA therapy (median 52.1% vs 46.4% without DARA, p=0.486) or prior exposure to DARA (92.1% vs 91.2%, p=0.966) were not associated with D50 NAb titers. Generalized linear models were used for evaluation of multiple factors associated with D50 NAb titers: at least 3 months since the last dose of anticlonal therapy (p<0.001), lymphocyte counts (p=0.001) and serum albumin levels at the time of vaccination (p=0.020) were independent predictors of NAb titers on D50. When seroconversion was defined as a NAb titer ≥50% at D50, then >3 months of treatment-free interva (HR:7.75, p<0.001,) was the strongest factor associated with seconversion. In conclusion, patients with AL amyloidosis have an attenuated response to vaccination with BNT162b2 especially among those on active therapy or with less than 3 months since the last dose of treatment. For such patients, an anamnestic dosing strategy could be considered, especially after completion of anticlonal therapy. [Formula presented] Disclosures: Kastritis: Genesis Pharma: Honoraria;Janssen: Consultancy, Honoraria, Research Funding;Takeda: Honoraria;Pfizer: Consultancy, Honoraria, Research Funding;Amgen: Consultancy, Honoraria, Research Funding. Terpos: Novartis: Honoraria;Janssen: Consultancy, Honoraria, Research Funding;Genesis: Consultancy, Honoraria, Research Funding;Celgene: Consultancy, Honoraria, Research Funding;BMS: Honoraria;Amgen: Consultancy, Honoraria, Research Funding;Takeda: Consultancy, Honoraria, Research Funding;Sanofi: Consultancy, Honoraria, Research Funding;GSK: Honoraria, Research Funding. Gavriatopoulou: Sanofi: Honoraria;GSK: Honoraria;Karyopharm: Honoraria;Takeda: Honoraria;Genesis: Honoraria;Janssen: Honoraria;Amgen: Honoraria. Dimopoulos: Amgen: Honoraria;BMS: Honoraria;Janssen: Honoraria;Takeda: Honoraria;BeiGene: Honoraria.