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Bivalent COVID-19 vaccines that contain two mRNAs encoding Wuhan-1 and Omicron BA.4/5 spike proteins are successful in preventing infection from the original strain and Omicron variants, but the quality of adaptive immune responses is still not well documented. This study aims at characterizing adaptive immune responses to the bivalent booster vaccination in 46 healthy participants. Plasma and PBMC were collected prior and three weeks after bivalent booster. We measured anti-N, anti-S, and RBD IgM, IgA, IgG plasma titers against original, Omicron BA.1, and BA.5 variants (pending) as well as total anti-S IgG titers and surrogate Virus Neutralization capacity against the Alpha, Delta, and BA.1 variant. With spectral flow-cytometry we identified peripheral blood B-cells specific for the RBD of the S-protein of the original and BA.1 variants. T-cell-specific responses were assessed by cytokine release assay after stimulation with SARS-CoV-2 peptides from the original, BA.1, BA.4, and BA.5 variants (pending). Finally, we performed TRB and IGH repertoire studies on sorted CD4+, CD8+, CD19+ lymphocytes, to study breadth of SARS-CoV-2 specific clonotypes (pending). 27/46 participants were analyzed;9 had SARS-CoV-2 infection (COVID+), while 18 are infection naive (COVID-). In both groups, median time since last dose of SARS-CoV-2 vaccine (3rd or 4th) was 11 months. All subjects were positive for anti-S IgG prior to bivalent booster. The COVID + group displayed anti-S IgG pre-booster levels and neutralization against BA.1 higher than the COVID- group. Significant increase post-boost of total anti-S IgG and BA.1 neutralizing activity was detected in the COVID- but not in the COVID+ group;however, no difference in neutralization activity post-boost was detected between the two groups. Furthermore, the COVIDgroup showed significant increase in the frequency of CD19+ and CD27+ switched memory B-cells specific for BA.1 RBD in post-boost compared to pre-boost samples. However, post-boost frequencies of the same B-cells were higher in the COVID+ compared to the COVID- group. These preliminary findings confirm that among individual immunized with the original COVID-19 mRNAvaccine, prior COVID infection provides increased protection against SARS-CoV-2 variants. They also demonstrate that booster immunization with the bivalent vaccine induces robust adaptive immune responses against Omicron variant.[Formula presented][Formula presented]Copyright © 2023 Elsevier Inc.
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Introduction: Combination of daratumumab (Dara) and lenalidomide (Len) may enhance the function of teclistamab (Tec), potentially resulting in improved antimyeloma activity in a broader population. We present initial safety and efficacy data of Tec-Dara- Len combination in patients with multiple myeloma (MM) in a phase 1b study (MajesTEC-2;NCT04722146). Method(s): Eligible patients who received 1-3 prior lines of therapy (LOT), including a proteasome inhibitor and immune-modulatory drug, were given weekly doses of Tec (0.72-or- 1.5 mg/kg with step-up dosing) + Dara 1800 mg + Len 25 mg. Responses per International Myeloma Working Group criteria, adverse events (Aes) per CTCAE v5.0, and for cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) per ASTCT guidelines, were assessed. Result(s): 32 patients received Tec-Dara- Len (0.72 mg/kg, n = 13;1.5 mg/kg, n = 19). At data cut-off (11 July 2022), median follow-up (range) was 5.78 months (1.0-10.4) and median treatment duration was 4.98 months (0.10-10.35). Median age was 62 years (38-75);87.5% were male. Median prior LOT was 2 (1-3), 18.8% were refractory to Dara and 28.1% refractory to Len. CRS was most frequent AE (81.3% [n = 26], all grade 1/2), 95% occurred during cycle1. Median time to onset was 2 days (1-8), median duration was 2 days (1-22). No ICANS were reported. Frequent Aes (>=25.0% across both dose levels) were neutropenia (75.0% [n = 24];grade 3/4: 68.8% [n = 22]), fatigue (43.8% [n = 14];grade 3/4: 6.3% [n = 2]), diarrhoea (37.5% [n = 12];all grade 1/2), insomnia (31.3% [n = 10];grade 3/4: 3.1% [n = 1]), cough (28.1% [n = 9];all grade 1/2), hypophosphatemia (25.0% [n = 8];all grade 1/2), and pyrexia (25% [n = 8];grade 3/4: 6.3% [n = 2]). Febrile neutropenia frequency was 12.5% (n = 4). Infections occurred in 24 patients (75.0%;grade 3/4: 28.1% [n = 9]). Most common were upper respiratory infection (21.9% [n = 7]), COVID-19 (21.9% [n = 7]), and pneumonia (21.9% [n = 7]). Three (9.4%) had COVID-19 pneumonia. One (3.1%) discontinued due to COVID-19 infection and this patient subsequently died of this infection. Overall response rate (ORR, median follow-up) was 13/13 (8.61 months) at 0.72 mg/kg and 13/16 evaluable patients (less mature at 4.17 months) at 1.5 mg/kg. 12 patients attained very good/better partial response at 0.72 mg/kg dose, and response was not mature for 1.5 mg/kg group. Median time to first response was 1.0 month (0.7-2.0). Preliminary pharmacokinetic concentrations of Tec-Dara- Len were similar as seen with Tec monotherapy. Tec-Dara- Len- treatment led to proinflammatory cytokine production and T-cell activation. Conclusion(s): The combination of Tec-Dara- Len has no new safety signals beyond those seen with Tec or Dara-Len individually. Promising ORR supports the potential for this combination to have enhanced early disease control through the addition of Tec. These data warrant further investigation.
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The TG6002.03 trial is a dose-escalation phase 1 clinical trial of TG6002 infusion via the hepatic artery in patients with liver-dominant colorectal cancer metastases. TG6002 is an engineered Copenhagen strain oncolytic Vaccinia virus, deleted of thymidine kinase and ribonucleotide reductase to enhance tumor selective viral replication and expressing FCU1, an enzyme converting the non-cytotoxic prodrug 5-fluorocytosine (5-FC) into the chemotherapeutic compound 5-fluorouracil (5-FU). In this trial, patients with advanced unresectable liver-dominant metastatic colorectal cancer who had failed previous oxaliplatin and irinotecan-based chemotherapy were treated with up to 2 cycles of TG6002 infusion 6 weeks apart via the hepatic artery on day 1 combined with oral 5-FC on days 5 to 14 (where day 1 = TG6002 infusion). TG6002 infusion was performed over 30 minutes via selective catheterization of the hepatic artery proper. 5-FC oral dosing was 50mg/kg x4 daily. Blood was sampled for TG6002 pharmacokinetics and 5-FC and 5-FU measurements. Sampling of liver metastases was performed at screening and on day 4 or day 8 for virus detection and 5-FC and 5-FU quantification. In total, 15 patients (median age 61 years, range 37-78) were treated in 1 UK centre and 2 centres in France and received a dose of TG6002 of 1 x 106 (n=3), 1 x 107 (n=3), 1 x 108 (n=3), or 1 x 109 pfu (n=6). Fourteen of the 15 patients received a single cycle of treatment, including one patient who did not received 5-FC, and one patient received two cycles. TG6002 was transiently detected in plasma following administration, suggesting a strong tissue selectivity for viral replication. In the highest dose cohort, a virus rebound was observed on day 8, concordant with replication time of the virus. In serum samples, 5-FU was present on day 8 in all patients with a high variability ranging from 0.8 to 1072 ng/mL and was measurable over several days after initiation of therapy. Seven of the 9 patients evaluable showed the biodistribution of the virus in liver lesions by PCR testing on day 4 or day 8. Translational blood samples showed evidence for T-cell activation and immune checkpoint receptor-ligand expression. At 1 x 109 pfu, there was evidence for T-cell proliferation and activation against tumour-associated antigens by ELISpot and for immunogenic cell death. In terms of safety, a total of 34 TG6002-related adverse events were reported, of which 32 were grade 1-2 and 2 were grade 3. The maximum tolerated dose was not reached, and a single dose-limiting toxicity was observed consisting of a myocardial infarction in a context of recent Covid-19 infection in a 78-year-old patient. These results indicate that TG6002 infused via the hepatic artery in combination with oral 5-FC was well tolerated, effectively localized and replicated in the tumor tissues, expressed its therapeutic payload and showed anti-tumoral immunological activity.
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Background & Aim: Amniotic fluid (AF)-derived EVs are currently under investigation for use as anti-inflammatory therapeutics in COVID-19 and COVID-19 long haulers. The dysregulation of the immune response induced by SARS-COV-2 is a key driver of both acute COVID-19 induced lung injury and long term COVID-19 sequela. There is a clear need to identify therapeutics that suppress excessive inflammation and reduce immune cell exhaustion to improve patient short term and long-term outcomes. Amniotic fluid (AF)- derived extracellular vesicles (EVs) have previously been shown to deliver anti-inflammatory and immune-modulatory signals to diverse cellular targets. We aimed to test if AF-EVs carry immune-suppressive molecules and can suppress T-cell immune activation and exhaustion in vitro. Methods, Results & Conclusion(s): The AF-EV biologic tested was derived from AF collected from consenting donors during planned, fullterm cesarean sections. AF was centrifuged and filtered to remove cellular debris and create a product containing AF-EVs and soluble extracellular components. Fluorescent EXODOT analysis was performed to demonstrate the presence of EV markers CD9, CD81, ALIX, and immune suppressive molecule PD-L1. T-cell activation/exhaustion was induced in vitro by treating human peripheral blood mononuclear cells with activation agent PHA for 3 days with the addition of AF-EVs or saline control. Immune activation/exhaustion was measured by flow cytometry to determine the expression of PD-1 on CD3+ T-cells. The AF-EV biologic was characterized to contain EVs with positive expression of CD9, CD81, ALIX, and PL-L1. T-cell activation/exhaustion was upregulated in response to PHA and was significantly reduced by 8% in AF-EV treated T-cells compared to saline control (77.7% vs 85.7%, respectively P<0.05). These findings demonstrate that AF-EVs do express PD-L1, a surface marker that has previously been demonstrated to contribute to exosome-mediated immunosuppression. Furthermore, we confirmed in vitro that AF-EVs suppress T-cell activation/ exhaustion in the presence of a T-cell activation agent. COVID-19 long haulers have been described to have upregulated and pro-longed immune activation and T-cell exhaustion, marked by an increase in PD1+ T-cells. Therefore, this finding serves as a starting point for the development of a potential mechanism of action that may describe AF-EV's therapeutic effect in COVID-19 long hauler patients.Copyright © 2023 International Society for Cell & Gene Therapy
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Introduction: Type 1 interferon (IFN) autoantibodies, such as anti-IFNalpha, have pathogenic significance in life-threatening COVID-19 pneumonia. Ten to twenty percent of severe COVID cases are associated with type I IFN autoantibodies. These autoantibodies likely pre-exist while others arise de novo relative to SARS-CoV-2 infection. It is unclear to what extent type I anti-IFN autoantibodies are induced by SARS-CoV-2 infection and contribute to COVID-19 severity. We investigated these phenomena in those with inborn errors of immunity (IEI) and rheumatic disease (RHE). Aim(s): We aim to compare the prevalence and neutralization ability of anti-IFNalpha autoantibodies in IEI and RHE patients using archived blood samples before and after the COVID-19 pandemic began. Method(s): We determined the presence of autoantibodies against IFNalpha in plasma samples by enzyme linked immunosorbent assay in 453 patients with IEI or RHE who were testing either before or after the COVID-19 pandemic began in March 2020. Using flow cytometry, we determined the function of IFNalpha autoantibodies in plasma to block CD4T cell activation by inhibiting STAT-1 phosphorylation. Result(s): We found that 25 patients with IEI or RHE were positive for anti-IFNalpha autoantibodies. 10 out of 229 patient samples collected before the pandemic (4.2%) tested positive whereas 15 out of 224 patient samples collected after the pandemic began (7.0%) were positive. Seven of the 25 patients (28%) who tested positive had neutralizing antibodies in plasma, which prevented STAT-1 phosphorylation in CD4T cells;all of these patients had partial recombination activating gene deficiency (pRD) except for one patient with autoimmunity, leukemia and selective IgA deficiency. One pRD patient had anti-IFNalpha autoantibodies with neutralization capacity before the pandemic, which persisted after hematopoietic stem cell transplantation (HSCT) with full immune reconstitution. The patient was immunized for SARS-CoV-2 before and after HSCT and acquired COVID-19 infection a year after HSCT. The patient was symptomatic but never hospitalized and fully recovered despite having anti-IFNalpha autoantibodies. Conclusion(s): Anti-IFNalpha autoantibody levels were comparable before and after the start of the COVID-19 pandemic in IEI and RHE patients but only 28% of cases were neutralizing. The clinical implications of these autoantibodies are yet to be determined.Copyright © 2023 Elsevier Inc.
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Background: The rapid development of SARS-CoV-2 mRNA vaccines has been a remarkable success of the COVID-19 pandemic, but vaccine-induced immunity is heterogeneous in immunocompromised populations. We sought to determine the immunogenicity of SARS-CoV-2 mRNA vaccines in a cohort of people with idiopathic CD4 lymphopenia (ICL). Method(s): 25-patients with ICL followed at the National Institutes of Health on a natural history protocol were evaluated between 2020-2022. Blood and serum was collected within 4-12 weeks after their second and/or third SARS-CoV-2 mRNA vaccine dose. Twenty-three matched healthy volunteers (HVs) provided blood samples at similar timepoints post-mRNA vaccination on a separate clinical protocol. Pre-vaccine blood samples were also used when available. Anti-spike and anti-receptor binding domain antibodies were measured. T-cell stimulation assays were performed to quantify SARS-CoV-2 specific T-cell responses. Comparisons were made with Wilcoxon test. Result(s): Twenty-participants with ICL had samples collected after their second mRNA vaccine and 7-individuals after the third dose. Median age at vaccination was 51-years (IQR: 44-62) and 12 were women (48%). Median CD4 T-cell count was 150 cells/muL (IQR: 85-188) at the time of vaccination, and 11-individuals (44%) had a baseline CD4 count <=100 cells/muL. HVs had a median age of 54-years (IQR: 43-60) with 13-women (56.5%). Anti-spike IgG antibody levels were significantly greater in HVs than those with ICL after 2-doses. Lower SARS-CoV-2 IgG antibody production was primarily observed in those with baseline CD4 T-cells <=100 cells/mul (Figure-1A). The decreased production in ICL remained after a third vaccine dose (Figure-1B). There was a significant correlation between anti-spike IgG and baseline CD4 count. Spike-specific CD4 T-cell responses in volunteers compared to those with ICL demonstrated similar levels of activation induced markers (CD154+CD69+) and cytokine production (IFNgamma+, TNFalpha+, IL2+) after two or three mRNA vaccine doses. Quantitatively the smallest responses were observed in those with lower baseline CD4 T-cells (Figure 1C-D). Minimal SARS-CoV-2 CD8 T-cell responses were detected in both groups. Conclusion(s): Patients with ICL and baseline CD4 T-cells >100 mount similar humoral and cellular immune responses to SARS-CoV-2 vaccination as healthy volunteers. Those with baseline CD4 T-cells <=100 have impaired vaccine- induced immunity and should be prioritized to additional boosters and continue other risk mitigation strategies. (Figure Presented).
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Assessment of viral load levels in various biological samples taken from the respiratory tract can be an indicator of an ongoing process of active viral replication and may be used to monitor severe respiratory viral infections. The study of the relationship between SARS-CoV-2 viral load and immunological laboratory parameters is an important step in the search for clinical markers of COVID-19. The aim of this research was to quantify viral load in patients with COVID-19 and to identify the relationship between viral load and changes in the parameters of the cellular component of the immune system. A laboratory examination was carried out on 74 patients diagnosed with COVID-19, they were divided into 3 groups based on the severity of the disease: mild, moderate, severe. Total viral load in clinical samples was determined by the number of SARS-CoV-2 RNA copies per 100 copies of the reference RNaseP gene. A comprehensive assessment of the cellular component of the immune system was performed using flow cytometry and direct monoclonal antibodies, and the IL-6, and C-reactive protein concentrations were determined. We revealed a relationship between the development of serious clinical conditions in the patients with COVID-19, and the levels of viral load. High levels of viral RNA in biological samples correlate with main indicators of the T cell component of the immune system associated with disease severity. In a subgroup of patients with an extremely high viral load, strong positive correlations were found between the relative numbers of cytotoxic lymphocytes (CD3+CD8+), activated T lymphocytes (CD3+HLA-DR+), as well as absolute and relative numbers of activated B lymphocytes and NK cells (CD3-CD25+). Laboratory monitoring of the cellular component of the immune system, along with the assessment of viral loads, should improve early assessment of clinical condition in the patients with COVID-19. Changes in expression levels of activation markers on immune cells can be potentially viewed as indicators of recovery during COVID-19.Copyright © Nikitin Yu.V. et al., 2023 The article can be used under the Creative Commons Attribution 4.0 License.
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Background: Better understanding of host inflammatory changes that precede development of severe COVID-19 could improve delivery of available antiviral and immunomodulatory therapies, and provide insights for the development of new therapies. Method(s): In plasma from individuals with COVID-19, sampled <=10 days from symptom onset from the All-Ireland Infectious Diseases Cohort study, we measured 61 biomarkers, including markers of innate immune and T cell activation, coagulation, tissue repair, lung injury, and immune regulation. We used principal component analysis (PCA) and k-means clustering to derive biomarker clusters, and univariate and multivariate ordinal logistic regression to explore association between cluster membership and maximal disease severity, adjusting for risk factors for severe COVID-19, including age, sex, ethnicity, BMI, hypertension and diabetes. Result(s): From March 2020-April 2021, we included 312 individuals, (median (IQR) age 62 (48-77) years, 7 (4-9) days from symptom onset, 54% male) in the analysis. PCA and clustering derived 4 clusters. Compared to cluster 1, clusters 2-4 were significantly older and of higher BMI but there were no significant differences in sex or ethnicity. Cluster 1 had low levels of inflammation, cluster 2 had higher levels of markers of tissue repair and endothelial activation (EGF, VEGF, PDGF, TGFalpha, serpin E1 and p-selectin). Cluster 3 and 4 were both characterised by higher overall inflammation, but compared to cluster 4, cluster 3 had downregulation of growth factors, markers of endothelial activation, and immune regulation (IL10, PDL1), but higher alveolar epithelial injury markers (RAGE, ST2). In univariate analysis, compared to cluster 1, cluster 3 had the highest odds of severe disease (OR (95% CI) 9.02 (4.62-18.31), followed by cluster 4: 5.59 (2.75-11.72) then cluster 2: 4.5 (2.38-8.81), all p < 0.05). Cluster 3 remained most strongly associated with severe disease in fully adjusted analyses;cluster 3: OR(95% CI) 5.99 (2.69-13.35), cluster 2: 3.14 (1.54-6.42), cluster 4: 3.13 (1.36-7.19), all p< 0.05). Conclusion(s): Distinct early inflammatory profiles predicted maximal disease severity independent of known risk factors for severe COVID-19. A cluster characterised by downregulation of growth factor and endothelial markers and early evidence of alveolar injury was associated with highest risk of developing severe COVID19. Whether this reflects a dysregulated inflammatory response that could improve targeted treatment requires further study. Heatmap of biomarker derived clusters and forest plot of association between clusters and disease severity. A: Heatmap demonstrating differences in biomarkers between clusters B: Forest plot demonstrating odds ratio of specific clusters for progressing to moderate or severe disease (reference Cluster 1), calculated using ordinal logistic regression. Odds ratio (95% CI) presented as unadjusted and fully adjusted (for age, sex, ethnicity, BMI, hypertension, diabetes, immunosuppression, smoking and baseline anticoagulant use). Maximal disease severity graded per the WHO severity scale.
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Background: Secondary lymphoid organs provide the adequate microenvironment for the development of antigen (Ag)-specific immune responses. The tight collaboration between CD4+ T cells and B cells in germinal centers is crucial to shape B cell fate and optimize antibody maturation. Dissecting these immune interactions remains challenging in humans, and animal models do not always recapitulate human physiology. To address this issue, we developed an in vitro 3D model of a human lymphoid organ. The model relies on a microfluidic device, enabling primary human cells to self-organize in an extracellular matrix (ECM) under continuous fluid perfusion. We applied this Lymphoid Organ-Chip (LO chip) system to the analysis of B cell recall responses to SARS-CoV-2 antigens. Method(s): We used a two-channel microfluidic Chip S1 from Emulate, where the top channel is perfused with antigen (spike protein or SARS-CoV-2 mRNA vaccine), while the bottom channel contains PBMC (n = 14 independent donors) seeded at high-density in a collagen-based ECM. Immune cell division and cluster formation were monitored by confocal imaging, plasmablast differentiation and spike-specific B cell amplification by flow cytometry, antibody secretion by a cell-based binding assay (S-flow). Result(s): Chip perfusion with the SARS-CoV-2 spike protein for 6 days resulted in the induction CD38hiCD27hi plasmablast maturation compared to an irrelevant BSA protein (P< 0.0001). Using fluorescent spike as a probe, we observed a strong amplification of spike-specific B cell (from 3.7 to 140-fold increase). In line with this rapid memory B cell response, spike-specific antibodies production could be detected as early as day 6 of culture. Spike perfusion also induced CD4+ T cell activation (CD38+ ICOS+), which correlated with the level of B cell maturation. The magnitude of specific B cell amplification in the LO chip was higher than in 2D and 3D static cultures at day 6, showing the added value of 3D perfused culture for the induction of recall responses. Interestingly, the perfusion of mRNA-based SARS-CoV-2 vaccines also led to strong B cell maturation and specific B cell amplification, indicating that mRNA-derived spike could be expressed and efficiently presented in the LO chip. Conclusion(s): We developed a versatile Lymphoid Organ-Chip model suitable for the rapid evaluation of B cell recall responses. The model is responsive to protein and mRNA-encoded antigens, highlighting its potential in the evaluation of SARS-CoV-2 vaccine boosting strategies.
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Background: Antigen-driven CD4+ T cell proliferation is a proposed mechanism of HIV-1 reservoir persistence. We previously reported that SARSCoV- 2 infection leads to increased detectable low-level HIV-1 plasm RNA blips months after COVID-19, but the impact of SARS-CoV-2-mediated T cell activation on expansion of HIV-1 reservoirs is not known. We sought to identify if SARSCoV- 2 infection leads to expansion of preferentially HIV-infected CD4+ T cells in people with HIV (PWH) on ART. Method(s): Five PWH with samples collected prior to and approximately two months after SARS-CoV-2 infection were identified. We performed a surface activation induced marker (AIM) assay using a CD4-optimized overlapping SARS-CoV-2 peptide pool to measure OX40/CD137 expression following peptide stimulation and sorted CD4+ T cells based on surface marker expression. ddPCR quantification of genomic HIV-1 DNA was performed on sorted subsets. Result(s): We observed an increase in the frequency of SARS-CoV-2 AIM+ non-naive CD4+ T cells following COVID-19 in samples from 4 of 5 participants (mean AIM+ % 0.13 pre- vs 0.31 post). A large percentage of non-naive AIM+ CD4+ T cells expressed PD1 compared with total non-naive cells before (76% vs 36%) and after (65% vs 19%) COVID-19;PD1 expression was lower following SARS-CoV-2 in both AIM+ and AIM- CD4+ T cell subsets (although very few cells were AIM+ prior to COVID-19). HIV-1 DNA levels in non-naive AIM- CD4+ T cells prior to COVID-19 unexpectedly decreased following infection (mean 3,522 to 766 copies/106 cells). The numbers of AIM+ cells obtained by cell sorting were overall low ( 3,863 mean) and only one participant had detectable DNA in post-COVID AIM+ CD4+ T cells. However, a large majority of this participant's post-COVID AIM+ cells harbored HIV-1 DNA (0.89 copies per cell) whereas HIV DNA in their AIM- cells decreased from 8,387 to not detected following SARSCoV- 2 infection. No HIV-1 DNA was detected in the small number of AIM+ cells obtained prior to COVID-19 in this participant. Conclusion(s): COVID-19 in PWH led to a modest SARS-CoV-2-specific CD4+ cell response approximately two months following acute presentation. One participant may have preferentially expanded HIV-1-infected, SARS-CoV-2- specific CD4+ T cells following COVID-19 but studies involving larger numbers of participants and larger numbers of cells will be needed to fully understand the impact of SARS-CoV-2 on clonal expansion and HIV persistence.
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Background: Current COVID-19 vaccines provide substantial protection against severe COVID-19, but they do not completely eliminate subsequent SARS-CoV-2 infections. We examined incidence of and immune differences against related but different common cold coronaviruses (ccCoV) as a proxy for response against a future emerging CoV among those with SARS-CoV-2 infection, COVID-19 vaccination, or neither exposure. Method(s): We assessed incidence of ccCoV (229E, HKU1, NL63, OC43) and rhinovirus/enterovirus infections among those with documented prior SARSCoV- 2 infection (n=493), prior COVID-19 vaccine, but no SARS-CoV-2 infection (n=1,568), or no prior SARS-CoV-2 infection or vaccination (n=2,874). We conducted a retrospective review of all individuals at Boston Medical Center that underwent a comprehensive respiratory panel polymerase chain reaction (CRP-PCR) test from November 30, 2020 to October 8, 2021 to estimate infection incidence. A subset within each group was assessed for coronavirus specific humoral and cellular immune responses, via pseudovirus neutralization and peptide stimulation T cell assays. Comparisons among the three groups were done using Chi-square and multi-variate Cox-proportional hazards models. Result(s): Incidence of symptomatic ccCoV was lower in those individuals with documented prior SARS-CoV-2 infection (1.0%) compared to those with COVID-19 vaccination (2.9%) or no prior SARS-CoV-2 exposure (1.8%, p = 0.01). Rhinovirus/enterovirus incidence was similar in all three groups (range 6.2 - 8.7%). Individuals with prior SARS-CoV-2 infection and those with previous COVID-19 vaccination had similar plasma neutralization against SARS-CoV-2, OC43, and 229E spike bearing pseudoviruses. SARS-CoV-2 (p = 0.01) and OC43 nucleocapsid (p = 0.02), but not spike specific peptides, yielded higher T cell responses in individuals with a prior SARS-CoV-2 infection as compared to those with COVID-19 vaccination or no prior SARS-CoV-2 exposure. Conclusion(s): Prior SARS-CoV-2 infection, but not COVID-19 vaccination, protects against subsequent related but different ccCoV symptomatic infection. This protection against symptomatic ccCoVs may be mediated by cellular responses to non-spike proteins. Future pan-coronavirus vaccines could be improved by including both spike and non-spike antigens.
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Background: Severe COVID-19 outcomes have been reported in people living with HIV (PLWH). High SARS-CoV-2 RNAemia has emerged as a hallmark of severe COVID-19, yet its pathogenic role in the context of COVID-19 in PLWH is currently unknown. We hereby measured SARS-CoV-2 RNAemia and explored its association with T-cell/humoral responses and clinical severity in PLWH. Method(s): Unvaccinated PLWH and age/sex-matched people living without HIV (PLWOH) hospitalized for radiologically-confirmed COVID-19 pneumonia were consecutively enrolled (March 2020-January 2021). We measured: SARS-CoV-2 RNAemia (RT-qPCR);T-cell activation (HLA-DR+CD-38+), cytotoxic T-cells [granzyme-B(GRZB)+perforin(PRF)+], GRZB/PRF production (MFI) by cytotoxic T-cells (flow cytometry);SARS-CoV-2-specific cytokines (IFN-gamma/ TNF-alpha/IL-2/IL-4/IL-17A)-producing T-cells, after SARS-CoV-2 spike peptides challenge (flow cytometry);anti-RBD antibodies (ELISA), Spike-ACE2 binding inhibition (receptor binding inhibition assay). Statistics: Mann-Whitney test and Spearman's correlation. Result(s): 18 PLWH (16 on cART;median CD4 361.5/mL;HIV-RNA< 50 cp/ mL in 15/18) and 18 PLWOH were included at a median of 10 days from symptoms onset (Fig.1A). PLWH had lower PaO2/FiO2 [140 (122-151.5) vs. 207 (156.3-309.3);P=0.0005] and higher SARS-CoV-2 RNAemia (Fig.1B). While humoral responses were comparable between groups ( Fig.1C-D), as was T-cell activation, PLWH showed skewed T-cell responses: higher perforin production by cytotoxic T-cells (Fig.1E);fewer SARS-CoV-2-specific IFN-gamma+ and IL-4+ CD4 T-cells (Fig.1F);lower Th1 tri-functional (IFN-gamma+TNF-alpha+IL-2+) and bi-functional (IFN-gamma+TNF-alpha+) CD4 T-cells (Fig.1G);reduced TNF-alpha+ CD8 T-cells (Fig.1H). Interestingly, SARS-CoV-2 RNAemia correlated negatively with PaO2/FiO2 nadir and SARS-CoV-2-specific T-cells, yet positively with perforin production by cytotoxic T-cells (Fig.1I-M). No correlations between RNAemia and humoral responses were found. Conclusion(s): As compared to HIV-uninfected patients, PLWH hospitalized for COVID-19 pneumonia feature high SARS-CoV-2 RNAemia which is linked to respiratory failure and skewed T-cell responses, with higher perforin production by cytotoxic T-cells, and yet fewer polyfunctional SARS-CoV-2-specific T-cells. Our data suggest a link between HIV-related T-cell dysfunction and poor control over circulating SARS-CoV-2 that may in turn influence COVID-19 severity in PLWH. (Figure Presented).
ABSTRACT
A strong link between COVID-19 severity and HLAC* 04:01 allele has been replicated in several Caucasian populations including Armenians. The results have led to an idea that HLA-C*04:01 may affect the immune response via three biological mechanisms: (i) disruption of the HLA-C mediated protection harnessing natural killer cells (NK);(ii) causing NK hypo-responsiveness through KIR2DL1;or (iii) over-activation and exhaustion of CTL and NK cells by stimulating functional KIR2DS4. To test those hypotheses, we re-analyzed HLA-genotypes and RNA-sequencing data of Overmyer et al. [Cell Systems 2021;12:23-40]. An ordinal regression of patients' status (i.e., non-COVID vs. COVIDnon- ICU vs. COVID-ICU) against HLA-C has corroborated the increase in the disease severity with increasing HLA-C*04:01 dosage (p< 0.003). DESeq2 analyses of the transcriptome (16444 loci) within COVID subset mapped 3586 down-regulated and 4031 up-regulated loci to the disease severity at FDR p<0.05. The results of enrichment analyses of those 7617 genes indicated aberrations in processes, such as T cell activation, inflammatory response, positive regulation of both NK-mediated cytotoxicity and interferon-gamma production. However, only 563 down- and 341 up-regulated loci had nominally associated with the HLA-C*04:01 carriage, reflecting its genetic association with severe symptoms. Using GTEx data and rs5010528 as proxy for HLAC* 04:01 (R2 = 0.97, 1kG EUR cohort), we found that HLA-C*04:01 was associated with multiple tissue (e.g., lung, heart and blood) RNA expressional and splicing changes in >10 protein-coding loci situated close to HLA-C. The ontology analysis of the loci implicated HLA-C*04:01 in altering antigen processing and presentation of endogenous peptide antigen via HLA class I via ER pathway (FDR p<0.0001), protection from NKmediated cytotoxicity (p<0.004), and innate immune response to other organisms (p<0.009). The work was supported by the Science Committee of RA (grant E17).
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Background: Belarus started developing a vaccine against SARS-CoV- 2 in 2021. The aim of the first stage of investigation was to evaluate the immunogenicity of the vaccine prototypes (VP) in vitro. Method(s): SARS-CoV- 2 strains (n = 7) were isolated using Vero E6 cells, inactivated with beta-propiolactone and purified. Antigens (Ag) were adsorbed on adjuvants: Al(OH)3 (Ag+AH group) or Al3PO4 (Ag+AP group). The single dose of VP (500 ul) was composed of 10 mug of Ag adsorbed on adjuvants (200 mug of Al3+). Blood samples from SARS-CoV- 2 recovered donors (n = 18) and healthy controls having no history of COVID-19 infection (n = 5) were used. Whole blood and Tag-it Violet labeled PBMCs were cultivated with VP, pure Ag, adjuvants (0.25-1 mug of Ag, 20 mug of Al3+ for probe) or pool of peptides, covering sequence of SARS-CoV- 2 N, S, M-proteins (PP), for 6 h and 7 days respectively. INF-gamma production and proliferation of CD3+ T-cells were assayed by FACS. Result(s): Counts of CD3+IFN-gamma+ T cells were 3.14(2.72-5.13)/ 1x105 CD3+ T cells in negative control (NC), and 12.73(10.09-33.95)/ 1x105 CD3+ T cells in specific positive control (PP) (n = 18, p < 0.0001), proving presence of antigen-specific T cells (ASCs) in donor blood. Samples were considered positive for VP and Ag immunogenicity when numbers of CD3+IFN-gamma+ T cells were 1.5 times greater compared with NC. Both VP types (Ag+AP, Ag+AH) and pure SARS-CoV- 2 isolates stimulated the production of INF-gamma by ASCs, responses ranged from 1 to 4 isolates of 7 studied per donor. Immunogenicity of Ag+AP, Ag+AH was confirmed by proliferation assay. Proliferation level was 1.07(0.97-2.38)% in Ag group with no differences from NC (n = 7, p > 0.5). Proliferation was significantly greater in VP groups compared with Ag: 2.47(1.65-2.68)% in Ag+AH, 4.03(2.56-4.61)% in Ag+AP (p = 0.009 and 0.002, respectively), stimulation of T cell was stronger by Ag+AP compared with Ag+AH (p = 0.009, M-U test). Pure adjuvants did not induce T cells response. There was no T-cell stimulation by Ag and VP in samples obtained from COVID-19 negative donors. Conclusion(s): The VP against SARS-CoV- 2 infection composed of inactivated virus adsorbed on Al(OH)3 and Al3PO4 adjuvants has immunogenic properties proven in two different immunological assays. VP stimulated activation and proliferation of ASCs in vitro suggesting this VP can be used for further preclinical in vivo evaluation.
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Background: Currently there are no published data regarding SARS-CoV-2 vaccine efficacy in paediatric-onset multiple sclerosis (MS) on disease-modifying treatments (DMTs). In adults, DMTs such as SIP-inhibitors (e.g., Fingolimod) and anti-CD20 therapies (e.g., Ocrelizumab) have been found to diminish SARS-CoV-2 vaccine efficacy. Objective(s): To ascertain whether young people with MS on DMTs generate a protective B-and/or T-cell response following SARS-CoV-2 vaccination or wild type (WT) infection. Method(s): Five millilitre additional blood was taken from MS patients during routine surveillance phlebotomy. Vaccination status and exposure to WT COVID was recorded. Serum samples measured SARS-CoV-2 antibodies using MSD quantitative assay. Multiplex T-cell stimulation assay was used to measure T-cell activity and T-cell response to SARS-CoV-2 Spike peptide was analysed. Result(s): Thirty-one MS patients (M:F 5:26;11-18 years) were included. DMTs included Ocrelizumab (n = 26);Fingolimod (n = 2);other DMT (n = 3). Vaccination status was confirmed in 12 children. 26/31 patients demonstrated protective B-cell responses, of which 10 were vaccinated +/- previous infection;six previously infected with unconfirmed vaccine status;and 10 had no infection/vaccine data available. Of the five patients with no B-cell response two were vaccinated. Six patients had impaired T-cell proliferation but 5/6 generated a B-cell response. T-cell proliferation upon exposure to Spike peptide was seen in 10 children'3/10 were vaccinated and 9/10 were being treated with Ocrelizumab. Patients on Fingolimod had impaired T-cell activity and no response to SARS-CoV-2 peptides, despite one being vaccinated and having a B-cell response. Conclusion(s): Fingolimod appeared to impair T-cell responses, however, only two patients were on this treatment. Those on Ocrelizumab have some protection following vaccination or WT exposure with 5/26 patients showing an impaired B-cell response but partial preservation of T-cell responses. While vaccination prior to starting DMT is ideal, some protection will be generated whilst on treatment.
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Background: Immune dysregulation plays a key role in determining COVID-19 disease severity. We aimed to analyze the T cell activation profile in COVID - 19 cases and its predictive role in disease severity and outcome. Material & methods: This was a prospective observational pilot study from a tertiary care COVID-19 hospital. Peripheral blood samples obtained between the fifth and seventh day of COVID-19 illness, were subjected to lymphocyte subset analysis using multicolor flowcytometry using a single tube, 8 antibodies (CD45, CD19, CD3, CD4, CD8, CD38, HLADR, and CD56) analysis. Correlation between lymphocyte subset analysis and clinical profile was determined. Results: 26 patients including 11 with mild disease and 15 with severe disease were enrolled. The median age was 58 years (range: 33-81), with a male: female ratio of 1.36:1. Significant lymphopenia was observed in the severe group compared to the mild group (p < 0.02). The absolute numbers of CD3+, CD4+, CD8 + T cells, B cells, and NK cells were significantly reduced in the severe group as compared to the mild group (p < 0.05). In patients with severe disease, the proportion of CD8 + and CD4 + T cells were significantly higher than those in patients with mild disease (p = 0.0372). Using ROC analysis, a CD4:8 T cell ratio of ≥ 2.63 and an activated (CD38 + HLA-DR+) CD8 T cell proportion of > 15.85% of the total CD8 T cell population, significantly determined the severe disease category. Conclusions: Severe COVID-19 is associated with severe lymphopenia, altered CD4/CD8 ratio and markedly increased CD8 T cell activation profile. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-022-01558-6.
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Purpose: SARS CoV-2 vaccination elicits both robust humoral and T-cell immune responses in healthy individuals. However, a comprehensive assessment of immune responses to SARS-CoV-2 vaccination in renal allograft recipients is variable and dependent primarily on Spike IgG levels. Here, we analyzed the humoral and T-cell responses in vaccinated transplant recipients. Method(s): 61Tx patients maintained either on Tacrolimus (TAC, 32) or Belatacept (BELA, 29) who were greater than one month post 2nd dose of the Pfizer BNT162b2, and 41 healthy individuals were enrolled. Fresh whole blood was incubated with SARS CoV-2 Spike peptides pool and the activated CD4+ (IL-2/TNF-alpha)+ and CD8+ (TNF-alpha/IFN-gamma)+ T cells were enumerated by flow cytometry and defined as CoV-2-specific T cells. Plasma was analyzed for Spike Receptor Binding Domain (RBD)-specific IgG by ELISA. The Spike RBD-specific IgG levels and Spikespecific CD4+/CD8+ T-cell immune responses were analyzed in TAC- and Bela- Tx patients along with healthy controls. Result(s): Our data demonstrated poor Spike IgG and T cell immune responses in Tx patients1M post-2nd dose of vaccine (21% v. 93% in positive Spike IgG and 37% v. 88% in positive T cell responses, Tx v. controls, respectively). However, 34% of Spike IgG (-) patients demonstrated positive CD4+ and/or CD8+ T-cell immune responses. No significant difference in T cell immunity was found between TAC and BELA treated patients. Conclusion(s): Immunocompromised Tx patients demonstrated significant defects in humoral and T cell immune response after vaccination. Patients maintained on TAC v. BELA demonstrated similar depressions in immune responses post-vaccination. 34% of vaccinated Tx patients, demonstrated Spike-specific T cell immunity despite being Spike IgG negative. This is suggestive of a divergent immune response with dominant cellular immunity. These observations are important since activation of T-cell immunity early after exposure to SARS-CoV2, while not preventing infection will likely modify severity of disease. (Table Presented).
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Purpose: Correlates of protection for SARS-CoV-2 vaccines are not well-established in kidney transplant recipients(KTRs). Studies have highlighted the importance of neutralizing antibodies(Abs), however data suggests T cell responses may play a secondary role in preventing reinfection. We performed a longitudinal assessment of immunogenicity, T and B cell response in KTRs following SARS-CoV-2 vaccination. Method(s): KTRs eligible for SARS-CoV-2 vaccination from 3/12/21 were enrolled. Baseline and weekly blood samples were collected for routine lab, SARS-CoV-2 spike protein Ab titers and cellular phenotyping for 12 weeks. Ab response was defined as a 10-fold increase in total binding IgG titers. To determine if T cell responses were induced by vaccination, we considered the proportion of activated non-naive CD4+ and CD8+ T cells post-vaccination. Result(s): 49 KTRs were enrolled ( Demographics -Table 1). 10 patients (20.4%) mounted an Ab response following vaccination. A history of COVID-19 was associated with an increased likelihood of developing an Ab response (OR: 18.3, 95% CI 3.2, 105.0, p=0.0005). For non-naive CD8+ T cells, a subset co-expressing CD38+Ki67+ was induced 1 week after the 1st immunization in some SARS-CoV- 2-naiive patients (P=0.12 versus P=0.14 for SARS-CoV-2-experienced adults, Fig 1A/B). For non-naive CD4+ T cells, induction of a subset co-expressing CD38+Ki67+ was observed at 1 week after the 1st immunization for SARS-CoV-2-naive participants (P = 0.09 for SARS-CoV-2-naive, P=0.03 for SARS-CoV-2-experienced adults, Fig 1C/D). For CD8+ and CD4+ T cells, dose 2 stimulated weak induction of the CD38+Ki67+ subset in the SARS-CoV-2-naive patients only (Fig 1A-D). Conclusion(s): Quantitative Ab responses were strongly associated with prior SARS-CoV-2 infection. Activated CD4+ and CD8+ T cell responses were evident in most patients irrespective of history of COVID-19. Further studies are needed to determine whether these activated CD4+ and CD8+ T cell responses were antigenspecific or confer immunity. (Table Presented).
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SESSION TITLE: Challenging Cases of Hemophagocytic Lymphohistiocytosis SESSION TYPE: Rapid Fire Case Reports PRESENTED ON: 10/19/2022 12:45 pm - 1:45 pm INTRODUCTION: Worsening respiratory disease is the most common complication of severe COVID-19. However, when patients develop multi-organ dysfunction, clinicians must have a high index of suspicion for rare syndromes such as hemophagocytic lymphohistiocytosis (HLH). CASE PRESENTATION: A 39-year-old male smoker presented with 1 week of shortness of breath and malaise. Initial physical examination revealed T 37.3 C, pulse 85 min-1, respiratory rate 18 breaths min-1, SPO2 96% and clear breath sounds without labored respirations. Chest X-ray showed bilateral patchy airspace opacities in the mid and lower lung fields. A SARS-COV2 PCR test was positive. The patient was prescribed antibiotics and discharged home. Subsequently, the patient's symptoms worsened and he presented 1 week later with SPO2 90% (O2 10 L/min via nasal cannula). He was admitted to the hospital with COVID-19 pneumonia and began remdesivir, barcitinib, systemic steroids, albuterol and IV antibiotics. On admission his complete blood count and complete metabolic panel were unremarkable. After 3 weeks of hospitalization, he developed multi-organ failure with acute liver injury, acute kidney injury, shock, pancytopenia and worsening hypoxemia leading to endotracheal intubation and mechanical ventilation. CT chest imaging showed bilateral ground glass opacities in the lungs with superimposed consolidation (figure 1). Blood cultures remained sterile, HIV, hepatitis B and C viral serologies were negative. Serum viral polymerase chain reaction detected Herpes Simplex Virus-1 (HSV-1) and Epstein Barr Virus (EBV) infections. Trans-jugular liver biopsy confirmed HSV-1 hepatitis and showed sub-massive hemorrhagic necrosis of the liver (figure 2). Bone marrow biopsy demonstrated phagocytic histiocytes engulfing red blood cells and platelets consistent with HLH (figure 3). The patient began HLH targeted therapy with anakinra and high dose steroids. Despite this, the patient continued to deteriorate, developed refractory shock and subsequently expired. DISCUSSION: HLH is a rare disease of the immune system in which a genetic or infectious trigger causes uncontrolled T cell activation. T cell activation triggers macrophage activation, cytokine storm and macrophage phagocytosis of erythrocytes, leukocytes, platelets and precursors in the bone marrow and other tissues. If the syndrome is unrecognized, it can quickly lead to multi-organ failure and death. EBV is the most common infectious trigger of HLH;however, infection with HSV-1 and SARS-COV-2 viruses have been identified as rare and independent causes. CONCLUSIONS: This case illustrates the high index of suspicion providers should have for HLH in patients with severe COVID-19 who develop multi-organ injuries. Once HLH is suspected, prompt initiation of HLH-94 protocol with etoposide and dexamethasone may be lifesaving. For those patients with liver failure, other agents (e.g. anakinra) may be provided. Reference #1: Ramos-Casals M, Brito-Zerón P, López-Guillermo A, et al.: Adult haemophagocytic syndrome. Lancet 2014;383:1503–1516 Reference #2: Risma K, Jordan MB: Hemophagocytic lymphohistiocytosis: updates and evolving concepts. Curr Opin Pediatr 2012;24:9–15 Reference #3: Trottestam H, Horne A, Aricò M, et al.: Chemoimmunotherapy for hemophagocytic lymphohistiocytosis: long-term results of the HLH-94 treatment protocol. Blood 2011;118:4577–4584 DISCLOSURES: No relevant relationships by Erin Biringen No relevant relationships by Christine Brennan No relevant relationships by Joann Hutto No relevant relationships by Daniel Puebla Neira