Enzyme-linked Immune Absorbent Spot Research Articles

Association of HBV serological markers with host antiviral immune response relevant hepatic inflammatory damage in chronic HBV infection.

The natural progression of chronic hepatitis B virus (HBV) infection is dynamic, but the longitudinal landscape of HBV serological markers with host antiviral immune response relevant hepatic inflammatory damage remains undetermined. To this issue, we studied the association of HBV serological markers with the severity of hepatic inflammatory damage and enumerated HBV-specific T cells using the cultured enzyme-linked immune absorbent spot (ELISpot). Five hundred and twenty-four treatment-naïve chronic HBV infection patients were enrolled. The Spearman correlation analysis revealed that in hepatitis B e antigen (HBeAg)-positive patients, all HBV virologic indicators negatively correlated with liver inflammatory damage and fibrosis (p < 0.01). Stronger correlations were accessed in the subgroup of HBeAg-positive patients with HBV DNA > 2 × 106 IU/mL (p < 0.01), whereas negative correlations disappeared in patients with HBV DNA ≤ 2 × 106 IU/mL. Surprisingly, in HBeAg-negative patients, the HBV DNA level was positively correlated with the hepatic inflammatory damage (p < 0.01). The relationship between type Ⅱ interferon genes expression and HBV DNA levels also revealed a direct shift from the initial negative to positive in HBeAg-positive patients with HBV DNA declined below 2 × 106 IU/mL. The number ofHBV-specific T cells were identified by interferon γ ELISpot assays and showed a significant increase from HBeAg-positive to HBeAg-negative group. The host's anti-HBV immunity remains effective in HBeAg-positive patients with HBV DNA levels exceeding 2 × 106 IU/mL, as it efficiently eliminates infected hepatocytes and inhibits HBV replication. However, albeit the increasing number of HBV-specific T cells, the host antiviral immune response shifts towards dysfunctional when the HBV DNA load drops below this threshold, which causes more pathological damage and disease progression.

Assessing Antigen-Specific T Cell Responses Through IFN-γ Enzyme-Linked Immune Absorbent Spot (ELISpot).

T cells are instrumental in protecting the host against invading pathogens and the development of cancer. To do so, they produce effector molecules such as granzymes, interleukins, interferons, and perforin. For the development and immunomonitoring of therapeutic applications such as cell-based therapies and vaccines, assessing T cell effector function is paramount. This can be achieved through various methods, such as 51Cr release assays, flow cytometry, and enzyme-linked immune absorbent spot (ELISpot) assays. For T cell ELISpots, plates are coated with antibodies directed against the effector molecule of interest (e.g., IFN-g). Subsequently, peripheral blood mononuclear cells (PBMCs) or isolated T cells are cultured on the plate together with stimuli of choice, and the production of effector molecules is visualized via labeled detection antibodies. For clinical studies, ELISpot is currently the gold standard to determine antigen-specific T cell frequencies. In contrast to 51Cr release assays, ELISpot allows for the exact enumeration of responding T cells, and compared to flow cytometry, ELISpot is more cost-effective and high throughput. Here, we optimize and describe, in a step-by-step fashion, how to perform a controlled IFN-γ ELISpot experiment to determine the frequency of responding or antigen-specific T cells in healthy human volunteers. Of note, this protocol can also be employed to assess the frequency of antigen-specific T cells induced in, e.g., vaccination studies or present in cellular products.

Multiparameter immunoprofiling for the diagnosis and differentiation of progressive versus nonprogressive nontuberculous mycobacterial lung disease-A pilot study.

Clinical prediction of nontuberculous mycobacteria lung disease (NTM-LD) progression remains challenging. We aimed to evaluate antigen-specific immunoprofiling utilizing flow cytometry (FC) of activation-induced markers (AIM) and IFN-γ enzyme-linked immune absorbent spot assay (ELISpot) accurately identifies patients with NTM-LD, and differentiate those with progressive from nonprogressive NTM-LD. A Prospective, single-center, and laboratory technician-blinded pilot study was conducted to evaluate the FC and ELISpot based immunoprofiling in patients with NTM-LD (n = 18) and controls (n = 22). Among 18 NTM-LD patients, 10 NTM-LD patients were classified into nonprogressive, and 8 as progressive NTM-LD based on clinical and radiological features. Peripheral blood mononuclear cells were collected from patients with NTM-LD and control subjects with negative QuantiFERON results. After stimulation with purified protein derivative (PPD), mycobacteria-specific peptide pools (MTB300, RD1-peptides), and control antigens, we performed IFN-γ ELISpot and FC AIM assays to access their diagnostic accuracies by receiver operating curve (ROC) analysis across study groups. Patients with NTM-LD had significantly higher percentage of CD4+/CD8+ T-cells co-expressing CD25+CD134+ in response to PPD stimulation, differentiating between NTM-LD and controls. Among patients with NTM-LD, there was a significant difference in CD25+CD134+ co-expression in MTB300-stimulated CD8+ T-cells (p <0.05; AUC-ROC = 0.831; Sensitivity = 75% [95% CI: 34.9-96.8]; Specificity = 90% [95% CI: 55.5-99.7]) between progressors and nonprogressors. Significant differences in the ratios of antigen-specific IFN-γ ELISpot responses were also seen for RD1-nil/PPD-nil and RD1-nil/anti-CD3-nil between patients with nonprogressive vs. progressive NTM-LD. Our results suggest that multiparameter immunoprofiling can accurately identify patients with NTM-LD and may identify patients at risk of disease progression. A larger longitudinal study is needed to further evaluate this novel immunoprofiling approach.

Abstract CT256: Modi-1, anti-citrullinated neoepitope vaccine alone and combined with checkpoint inhibitors in patients with head and neck, breast, renal and ovarian carcinoma: protocol for the ModiFY phase I/II basket clinical Ttial

Abstract Stressful conditions in the tumor microenvironment induce autophagy in cancer cells as a mechanism to promote their survival. High levels of calcium within autophagosomes activates peptidylarginine deaminase enzymes which convert arginine residues within polypeptides to citrulline and alters proteolytic cleavage. In the presence of inflammation, the MHC-II pathway presents these new citrullinated peptides to CD4 T cells. Modi-1 vaccine comprising three citrullinated, adjuvanted peptides induces and expands a population of activated CD4 T cells. On reaching the tumor site the CD4 T cells release proinflammatory cytokines including, INFγ, which upregulates MHC class II and the same, but endogenous, modified peptides are presented on the tumor cell surface. This likely causes a positive feed-forward loop with killing of tumor cells. The objective of this study is to evaluate the safety, tolerability, cellular immune and tumor response to 2 citrullinated vimentin and one citrullinated enolase peptide each conjugated to the toll-like receptor 1/2 adjuvant Amplivant® ModiFY is an open-label, prospective, multicohort, multicenter, phase I/II basket trial. Eligible patients have unresectable disease in one of the following tumor types: Squamous Cell Carcinoma of the Head and Neck (SCCHN), Triple Negative Breast Cancer, Renal Cell Carcinoma and High Grade Serous Ovarian Carcinoma. Depending on the status of the disease and eligibility for standard of care (SOC) checkpoint inhibitor (CPI) monotherapy, patients will be treated either with Modi-1 alone or Modi-1 +SOC CPI. A randomized neoadjuvant sub-study in patients with SCCHN scheduled to have tumor resection surgery is included in the protocol. These patients are randomized 1:1 to receive either Modi-1 alone or Modi-1+ pembrolizumab. The primary endpoints are the adverse event rate as measured by CTCAE v5.0 in the initial dose escalation cohorts and the strength of the cellular immune response IFNγ enzyme-linked immune absorbent spot (ELISpot) assay in the dose expansion cohorts. Secondary endpoints (RECIST 1.1 and iRECIST), are objective response rate duration of response, progression-free survival, and overall survival. In the SCCHN neoadjuvant cohort, tumor infiltrating lymphocytes in resected tumor tissue will be profiled using scRNAseq and immunohistochemistry. The study is an adaptive trial, comprising 3+3 dose escalation cohorts followed by a Simon 2-stage design in the dose expansion cohorts. The Modi-1 only dose expansion cohort will recruit 16 patients of each of the target tumor types, whilst the Modi-1+CPI will recruit a total of 21 patients. A total of 21/138 patients have been treated to date. ClinicalTrials.gov NCT05329532 Citation Format: Lindy G. Durrant, Fayaz Masters, Samantha Paston, Robert Miller, David J. Pinato, Rebecca Herbertson, Anne Armstrong, Stefan Symeonides, Christian Ottensmeier. Modi-1, anti-citrullinated neoepitope vaccine alone and combined with checkpoint inhibitors in patients with head and neck, breast, renal and ovarian carcinoma: protocol for the ModiFY phase I/II basket clinical Ttial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT256.

PS-BPB03-3: MODIFIED ANGIOTENSIN II PEPTIDE VACCINE FOR HYPERTENSION

We and others recently reported that an angiotensin II vaccine for hypertension successfully attenuated elevated blood pressures in an animal model without any immunogenic side effects. In this system, an immunogenic molecule (i.e., KLH) with adjuvants provides an antigen that supports the activation of helper T cells. We recently developed a novel peptide, AJP001, that possesses both a mouse T-cell epitope and adjuvant action. Direct conjugation to the antigen is useful for peptide vaccines without the addition of adjuvants. In this study, the efficacy of an angiotensin II and AJP001-conjugated peptide vaccine (AJP001- angiotensin II) was evaluated in mice. The anti-angiotensin II antibody titer was measured in Balb/C mice following three injections of AJP001-angiotensin II at 2-week intervals. SBP was measured during vaccination of Balb/C mice treated with angiotensin II infusion. AJ-angiotensin II treatment resulted in an increase in the anti-angiotensin II antibody titer in a dose-dependent manner without the addition of adjuvants. In the analysis of the humoral immune response, AJP001-angiotensin II mainly elicited IL-4 and IL-10 production, as measured by an enzyme-linked immune absorbent spot assay, which suggests the induction of a Th2 response. Importantly, cotreatment with purified antibodies attenuated angiotensin II-induced extracellular signal-regulated kinase phosphorylation in cultured vascular smooth muscle cells. The SBP in immunized mice was significantly lower than that in nonimmunized mice (135.9 ± 8.5 vs. 154.9 ± 16.8 mmHg, P = 0.02). Furthermore, angiotensin II-induced perivascular fibrosis in the heart was significantly attenuated in immunized mice, which also exhibited decreased mRNA expression of collagen I/III and transforming growth factor-beta. It may be a simple and useful therapeutic peptide vaccine, and we now further modify the T cell epitope for clinical trials.

611: HOST IMMUNE ENDOTYPING IN PEDIATRIC ACUTE RESPIRATORY FAILURE

Introduction: Acute respiratory failure (ARF) is a common reason for admission to the pediatric intensive care unit (PICU). The ability to immune endotype critically ill children with ARF to understand if adaptive or innate defects belie severity of illness, and then modulate these defects by evaluating therapies in an ex vivo fashion may be paradigm shifting. We have previously shown that T-cell IFN-γ (adaptive) and monocyte TNF-α (innate) production, as quantitated by the Enzyme-Linked Immune Absorbent Spot (ELISpot) assay, reflect immune competence in COVID-19 pneumonia, and illness severity is associated with decreased circulating lymphocytes with innate and adaptive immune suppression, evidenced by decreased IFN-ɣ and TNF-α production. This promising approach may have significant benefits in critically ill children, as it is unknown if specific measures of innate and adaptive immune function correlate with illness severity. We hypothesize that serial quantitative analysis of IFN-γ and TNF-α production by ELISpot will accurately reflect host immune status in critically ill children with ARF. Methods: In this IRB approved prospective cohort study, children admitted to the PICU with ARF had serial blood analysis using ELISpot for IFN-γ and TNF-α production after positive stimulus with CD3/28 and LPS respectively. ELISpot reporting for cytokine production is done as number of cells secreting the relevant cytokine; data was compared to relevant patient metadata and clinical outcomes. Results: We are currently enrolling patients, and have analyzed two patients with ARF secondary to pneumonia. Patient 1 had 19 cells secreting IFN-ɣ on day 1 and 69 cells on day 5; and 582 cells secreting TNF-α on day 1 and 1217 cells on day 5. Patient 2 had 18 cells secreting IFN-ɣ on day 1 and 144 cells on day 5; and 189 cells secreting TNF-α on day 1 and 466 cells on day 5. Conclusions: Preliminary analysis of IFN-γ and TNF-α production by ELISpot shows surprisingly more IFN-γ reduction early in disease course, which may be a driver of viral replication and severity of illness. Over 5 days, both patients had appropriately hyper inflammatory innate responses. With improved power and additional time points, there is potential to introduce adaptive stimulants early in disease trajectory that may alter clinical outcomes.

2158. Monitoring of Varicella specific T-cell mediated immunity in pediatric allogenic hematopoietic stem cell transplant recipients

Abstract Background Varicella-zoster virus (VZV) infection is known to occur in 13-55% of patients within the first year after hematopoietic stem cell transplant (HSCT). The dynamics of recovery of VZV-specific T cell- mediated immunity (CMI) and its role for prevention and control of VZV reactivation are rarely reported in pediatric allogenic HSCT recipients. Methods From April 2019 to February 2020, subjects aged younger than 19 years who underwent allogenic HSCT with at least 1 year of follow up at Asan Medical Center Children`s Hospital were prospectively enrolled. All of them underwent VZV-specific enzyme-linked immune absorbent spot (ELISPOT) assays just before HSCT and then at 1, 3 months following HSCT. Extension study, which measures VZV-specific CMI before and after VZV vaccination at 24 months post-HSCT, is currently underway. Results A total of 32 HSCT recipients with a median age of 11years were enrolled. 37.5% underwent haploidentical HSCT and VZV R+ were in 78.1%. Antiviral prophylaxis was applied to all HSCT recipients, of which 65.6% were applied up to 365 days after HSCT. During this study period, only 1 patient (3.1%) experienced herpes zoster at 23 months following HSCT, whose VZV-specific ELISPOT assay showed 0, 1, and 0 spot-forming cells (SFC)/2.0x105 cells before HSCT, 1 and 3 months following HSCT, respectively. The presence of VZV-specific CMI ≥ 1 SFC/ 2.0 × 105 cells was observed in 62.5 % before HSCT, 65.6 % at 1 month, 59.4 % at 3 months, and 70% at 24 months after HSCT, respectively. However, only 18.8 % ,12.5 % and 21.9 % recovered VZV-specific CMI with ≥ 5 SFC/ 2.0 × 105 cells at 1, 3 and 24 months following HSCT. Meanwhile, all the 4 recipients who got the 1st VZV vaccination at 24 months following HSCT showed recovery of VZV-specific CMI with ≥ 5 SFC/ 2.0 × 105 cell. Recovery of VZV-specific CMI after HSCT was not associated with VZV serostatus, chickenpox history, type of HSCT, conditioning regimens, and GVHD. Conclusion Only a few pediatric allogenic HSCT recipients recovered VZV-specific CMI with ≥ 5 SFC/ 2.0 × 105 cell up to 2 years following HSCT. In addition, the association between VZV-specific CMI and VZV reactivation could not be analyzed because no one experienced VZV reactivation within 1 year after HSCT. More long-term large-scale multicenter study is mandatory to supplement these parts. Disclosures All Authors: No reported disclosures.

2321. Long-term immunogenicity of Ad26.RSV.preF/RSV preF protein vaccine against RSV in a phase 2b study by age and risk level

Abstract Background Respiratory syncytial virus (RSV) can cause serious lower respiratory tract disease (LRTD) among older adults. There is no licensed RSV vaccine. In CYPRESS (a randomized, double-blind, placebo-controlled, phase 2b proof-of concept trial; NCT03982199), an Ad26.RSV.preF/RSV preF protein vaccine demonstrated 80.0% efficacy for prevention of RSV LRTD and 69.8% efficacy for prevention of any RSV acute respiratory infection in adults aged ≥65 years through the first RSV season. This study evaluated the durability of immune responses elicited by Ad26.RSV.preF/RSV preF protein after two RSV seasons (up to 1.5 years post-vaccination) in the overall study population and in groups of participants stratified by age and risk level for severe RSV LRTD. Methods Participants (N=5782) were randomized 1:1 to receive vaccine or placebo before the RSV season. The primary endpoint was first occurrence of RSV LRTD. RSV A2 virus neutralizing antibodies (VNAs; through Day 365), RSV preF binding antibodies (through Day 533), and RSV-F–specific IFN-γ enzyme-linked immune absorbent spot (ELISpot; through Day 533), were evaluated in an immunogenicity subset (n=195; ages 65–74 years: n=141; 75–84 years: n=47; ≥85 years: n=6; increased risk [chronic heart or lung disease]: n=48; non-increased risk: n=147). Results In the vaccine group of the immunogenicity subset, RSV A2 VNAs peaked at Day 15 and were maintained at 2.8-fold over baseline at 1 year. Similarly, RSV preF-specific binding antibodies peaked at Day 15 and were maintained at 2.1-fold above baseline at 1.5 years. Median RSV-F–specific IFN-γ T-cell frequency increased from 34 spot-forming cells (SFC)/106 peripheral blood mononuclear cells (PBMCs) at baseline to 143 SFC/106 PBMCs at 1.5 years. Comparable immune responses were observed in age/risk subgroups. No relevant changes were observed in the placebo group at any time point. Pre-existing Ad26 VNAs did not appear to impact RSV-specific immune response durability. Conclusion Ad26.RSV.preF/RSV preF protein vaccine was efficacious and elicited robust, durable (to at least 1.5 years) humoral and cellular immune responses in adults aged ≥65 years, older participants (≥75 years), and in participants with increased risk for severe RSV LRTD. Disclosures Christy A. Comeaux, MD, PhD, Janssen Vaccines &amp; Prevention B.V.: Employee Ann R. Falsey, MD, BioFire Diagnostics: Grant/Research Support|Janssen: Grant/Research Support|Merck Sharp &amp; Dohme: Grant/Research Support|Novavax: Advisor/Consultant|Pfizer: Grant/Research Support Kristi Williams, PhD, Janssen Research and Development: Employee John E. Ervin, MD, The Alliance for Multispecialty Research – KCM: Contractual agreement for conduct of study protocol Arangassery R. Bastian, PhD, Janssen Vaccines &amp; Prevention BV: Employee Joris Menten, PhD, Janssen Infectious Diseases: Employee Els De Paepe, MSc, Janssen Infectious Diseases: employee Sjouke Vandenberghe, PhD, Janssen Infectious Diseases: Employee Eric K. H. Chan, PhD, Janssen Global Services, LLC: Employee Jerald Sadoff, MD, Janssen Vaccines &amp; Prevention BV: Employee Macaya Douoguih, MD, MPH, Janssen Vaccines &amp; Prevention B.V.: Employee Benoit Callendret, PhD, Janssen Vaccines &amp; Prevention B.V.: Employee Esther Heijnen, MD, PhD, Janssen Vaccines &amp; Prevention B.V.: Employee.

Phenotypic and functional changes of T cell subsets after CoronaVac vaccination

BackgroundThe pandemic coronavirus disease 2019 (COVID-19) is a major global public health concern and several protective vaccines, or preventive/therapeutic approaches have been developed. Sinovac-CoronaVac, an inactivated whole virus vaccine, can protect against severe COVID-19 disease and hospitalization, but less is known whether it elicits long-term T cell responses and provides prolonged protection. MethodsThis is a longitudinal surveillance study of SARS-CoV-2 receptor binding domain (RBD)-specific IgG levels, neutralizing antibody levels (NAb), T cell subsets and activation, and memory B cells of 335 participants who received two doses of CoronaVac. SARS-CoV-2 RBD-specific IgG levels were measured by enzyme-linked immunosorbent assay (ELISA), while NAb were measured against two strains of SARS-CoV-2, the Wuhan and Delta variants. Activated T cells and subsets were identified by flow cytometry. Memory B and T cells were evaluated by enzyme-linked immune absorbent spot (ELISpot). FindingsTwo doses of CoronaVac elicited serum anti-RBD antibody response, elevated B cells with NAb capacity and CD4+ T cell-, but not CD8+ T cell-responses. Among the CD4+ T cells, CoronaVac activated mainly Th2 (CD4+ T) cells. Serum antibody levels significantly declined three months after the second dose. InterpretationCoronaVac mainly activated B cells but T cells, especially Th1 cells, were poorly activated. Activated T cells were mainly Th2 biased, demonstrating development of effector B cells but not long-lasting memory plasma cells. Taken together, these results suggest that protection with CoronaVac is short-lived and that a third booster dose of vaccine may improve protection.

SARS-CoV-2 Spike-specific IFN-γ T-cell Response After COVID-19 Vaccination in Patients With Chronic Kidney Disease, on Dialysis, or Living With a Kidney Transplant.

Background.Studies have shown that coronavirus disease 2019 (COVID-19) vaccination is associated with a lower humoral response in vulnerable kidney patients. Here, we investigated the T-cell response following COVID-19 vaccination in kidney patients compared with controls.Methods.Patients with chronic kidney disease (CKD) stage G4/5 [estimated glomerular filtration rate <30 mL/min/1.73 m2], on dialysis, or living with a kidney transplant and controls received 2 doses of the mRNA-1273 COVID-19 vaccine. Peripheral blood mononuclear cells were isolated at baseline and 28 d after the second vaccination. In 398 participants (50% of entire cohort; controls n = 95, CKD G4/5 n = 81, dialysis n = 78, kidney transplant recipients [KTRs] n = 144)‚ SARS-CoV-2-specific T cells were measured using an IFN-γ enzyme-linked immune absorbent spot assay.Results.A significantly lower SARS-CoV-2-specific T-cell response was observed after vaccination of patients on dialysis (54.5%) and KTRs (42.6%) in contrast to CDK G4/5 (70%) compared with controls (76%). The use of calcineurin inhibitors was associated with a low T-cell response in KTRs. In a subset of 20 KTRs, we observed waning of the cellular response 6 mo after the second vaccination, which was boosted to some extent after a third vaccination, although T-cell levels remained low.Conclusion.Our data suggest that vaccination is less effective in these patient groups, with humoral nonresponders also failing to mount an adequate cellular response, even after the third vaccination. Given the important role of T cells in protection against disease and cross-reactivity to SARS-CoV-2 variants, alternative vaccination strategies are urgently needed in these high-risk patient groups.

IFN-γ ELISpot in Severe Cutaneous Adverse Reactions to First-Line Antituberculosis Drugs in an HIV Endemic Setting

Severe cutaneous adverse reactions related to first-line antituberculosis drugs are associated with high mortality and long-term morbidity. Oral sequential drug challenge, as a form of drug provocation testing, helps to salvage therapy by identifying culprit drugs but is associated with risk and is costly. IFN-γ enzyme-linked immune absorbent spot (ELISpot), an adjunctive invitro diagnostic tool, may help to guide risk-stratification approaches. To determine the diagnostic accuracy of IFN-γ ELISpot against full-dose sequential drug challenge, we analyzed samples collected prospectively at multiple time points in 32 patients with first-line antituberculosis drug‒associated severe cutaneous adverse reaction (81% HIV infected, 25 with drug reaction with eosinophilia and systemic symptoms, and 7 with Stevens‒Johnson syndrome/toxic epidermal necrolysis). Sensitivity of IFN-γ ELISpot was 33% (4 of 12), 13% (1 of 8), 11% (1 of 9), and 0% (0 of 4) for rifampicin, isoniazid, pyrazinamide, and ethambutol, respectively (positivity threshold ≥50 spot forming units/million cells). Specificity was 100% for all the four drugs. Rifampicin IFN-γ ELISpot sensitivity increased to 58% (7 of 12) if a threshold of 20 spot forming units was used and to 75% (3 of 4) when restricted to samples <12 weeks after acute severe cutaneous adverse reaction event; specificity remained 100% for both. IFN-γ ELISpot offers adequate risk stratification of rifampicin severe cutaneous adverse reaction using acute samples and lowered threshold for positivity. Given the low sensitivity of IFN-γ ELISpot for other first-line antituberculosis drugs, additional optimization is needed to improve risk-stratification potential.

Immunogenicity and safety of COVID-19 vaccination in patients with primary Sjögren’s syndrome

ObjectivesTo evaluate humoral and cellular immune responses and adverse events (AEs) after COVID-19 vaccination in patients with primary Sjögren’s syndrome (pSS) compared to healthy controls (HC), and disease activity following...

SARS-CoV-2-specific antibody and T-cell responses 1 year after infection in people recovered from COVID-19: a longitudinal cohort study

BackgroundThe memory immune response is crucial for preventing reinfection or reducing disease severity. However, the robustness and functionality of the humoral and T-cell response to SARS-CoV-2 remains unknown 12 months after initial infection. The aim of this study is to investigate the durability and functionality of the humoral and T-cell response to the original SARS-CoV-2 strain and variants in recovered patients 12 months after infection.MethodsIn this longitudinal cohort study, we recruited participants who had recovered from COVID-19 and who were discharged from the Wuhan Research Center for Communicable Disease Diagnosis and Treatment at the Chinese Academy of Medical Sciences, Wuhan, China, between Jan 7 and May 29, 2020. Patients received a follow-up visit between Dec 16, 2020, and Jan 27, 2021. We evaluated the presence of IgM, IgA, and IgG antibodies against the SARS-CoV-2 nucleoprotein, Spike protein, and the receptor-binding domain 12 months after initial infection, using ELISA. Neutralising antibodies against the original SARS-CoV-2 strain, and the D614G, beta (B.1.351), and delta (B.1.617.2) variants were analysed using a microneutralisation assay in a subset of plasma samples. We analysed the magnitude and breadth of the SARS-CoV-2-specific memory T-cell responses using the interferon γ (IFNγ) enzyme-linked immune absorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) assay. The antibody response and T-cell response (ie, IFN-γ, interleukin-2 [IL-2], and tumour necrosis factor α [TNFα]) were analysed by age and disease severity. Antibody titres were also analysed according to sequelae symptoms.FindingsWe enrolled 1096 patients, including 289 (26·4%) patients with moderate initial disease, 734 (67·0%) with severe initial disease, and 73 (6·7%) with critical initial disease. Paired plasma samples were collected from 141 patients during the follow-up visits for the microneutralisation assay. PBMCs were collected from 92 of 141 individuals at the 12-month follow-up visit, of which 80 were analysed by ELISpot and 92 by ICS assay to detect the SARS-CoV-2-specific memory T-cell responses. N-IgG (899 [82·0%]), S-IgG (1043 [95·2%]), RBD-IgG (1032 [94·2%]), and neutralising (115 [81·6%] of 141) antibodies were detectable 12 months after initial infection in most individuals. Neutralising antibodies remained stable 6 and 12 months after initial infection in most individuals younger than 60 years. Multifunctional T-cell responses were detected for all SARS-CoV-2 viral proteins tested. There was no difference in the magnitude of T-cell responses or cytokine profiles in individuals with different symptom severity. Moreover, we evaluated both antibody and T-cell responses to the D614G, beta, and delta viral strains. The degree of reduced in-vitro neutralising antibody responses to the D614G and delta variants, but not to the beta variant, was associated with the neutralising antibody titres after SARS-CoV-2 infection. We also found poor neutralising antibody responses to the beta variant; 83 (72·2%) of 115 patients showed no response at all. Moreover, the neutralising antibody titre reduction of the recovered patient plasma against the delta variant was similar to that of the D614G variant and lower than that of the beta variant. By contrast, T-cell responses were cross-reactive to the beta variant in most individuals. Importantly, T-cell responses could be detected in all individuals who had lost the neutralising antibody response to SARS-CoV-2 12 months after the initial infection.InterpretationSARS-CoV-2-specific neutralising antibody and T-cell responses were retained 12 months after initial infection. Neutralising antibodies to the D614G, beta, and delta viral strains were reduced compared with those for the original strain, and were diminished in general. Memory T-cell responses to the original strain were not disrupted by new variants. This study suggests that cross-reactive SARS-CoV-2-specific T-cell responses could be particularly important in the protection against severe disease caused by variants of concern whereas neutralising antibody responses seem to reduce over time.FundingChinese Academy of Medical Sciences, National Natural Science Foundation, and UK Medical Research Council.

SARS-CoV-2-specific memory B cells can persist in the elderly who have lost detectable neutralizing antibodies.

Memory B cells (MBCs) can provide a recall response able to supplement waning antibodies (Abs) with an affinity-matured response better able to neutralize variant viruses. We studied a cohort of elderly care home residents and younger staff (median age of 87 years and 56 years, respectively), who had survived COVID-19 outbreaks with only mild or asymptomatic infection. The cohort was selected because of its high proportion of individuals who had lost neutralizing antibodies (nAbs), thus allowing us to specifically investigate the reserve immunity from SARS-CoV-2–specific MBCs in this setting. Class-switched spike and receptor-binding domain (RBD) tetramer–binding MBCs persisted 5 months after mild or asymptomatic SARS-CoV-2 infection, irrespective of age. The majority of spike- and RBD-specific MBCs had a classical phenotype, but we found that activated MBCs, indicating possible ongoing antigenic stimulation or inflammation, were expanded in the elderly group. Spike- and RBD-specific MBCs remained detectable in the majority of individuals who had lost nAbs, although at lower frequencies and with a reduced IgG/IgA isotype ratio. Functional spike-, S1 subunit of the spike protein– (S1-), and RBD-specific recall was also detectable by enzyme-linked immune absorbent spot (ELISPOT) assay in some individuals who had lost nAbs, but was significantly impaired in the elderly. Our findings demonstrate that a reserve of SARS-CoV-2–specific MBCs persists beyond the loss of nAbs but highlight the need for careful monitoring of functional defects in spike- and RBD-specific B cell immunity in the elderly.

BpOmpW Antigen Stimulates the Necessary Protective T-Cell Responses Against Melioidosis.

Melioidosis is a potentially fatal bacterial disease caused by Burkholderia pseudomallei and is estimated to cause 89,000 deaths per year in endemic areas of Southeast Asia and Northern Australia. People with diabetes mellitus are most at risk of melioidosis, with a 12-fold increased susceptibility for severe disease. Interferon gamma (IFN-γ) responses from CD4 and CD8 T cells, but also from natural killer (NK) and natural killer T (NKT) cells, are necessary to eliminate the pathogen. We previously reported that immunization with B. pseudomallei OmpW (BpOmpW antigen) protected mice from lethal B. pseudomallei challenge for up to 81 days. Elucidating the immune correlates of protection of the protective BpOmpW vaccine is an essential step prior to clinical trials. Thus, we immunized either non-insulin-resistant C57BL/6J mice or an insulin-resistant C57BL/6J mouse model of type 2 diabetes (T2D) with a single dose of BpOmpW. BpOmpW induced strong antibody responses, stimulated effector CD4+ and CD8+ T cells and CD4+ CD25+ Foxp3+ regulatory T cells, and produced higher IFN-γ responses in CD4+, CD8+, NK, and NKT cells in non-insulin-resistant mice. The T-cell responses of insulin-resistant mice to BpOmpW were comparable to those of non-insulin-resistant mice. In addition, as a precursor to its evaluation in human studies, humanized HLA-DR and HLA-DQ (human leukocyte antigen DR and DQ isotypes, respectively) transgenic mice elicited IFN-γ recall responses in an enzyme-linked immune absorbent spot (ELISpot)-based study. Moreover, human donor peripheral blood mononuclear cells (PBMCs) exposed to BpOmpW for 7 days showed T-cell proliferation. Finally, plasma from melioidosis survivors with diabetes recognized our BpOmpW vaccine antigen. Overall, the range of approaches used strongly indicated that BpOmpW elicits the necessary immune responses to combat melioidosis and bring this vaccine closer to clinical trials.

549 An RNA-lipoplex (RNA-LPX) vaccine demonstrates strong immunogenicity and promising clinical activity in a Phase I trial in cutaneous melanoma patients with no evidence of disease at trial inclusion

BackgroundLipo-MERIT is an ongoing, first-in-human, open-label, dose-escalation Phase I trial investigating safety, tolerability and immunogenicity of BNT111 in patients with advanced melanoma. BNT111 is an RNA-LPX vaccine targeting the melanoma tumor-associated antigens (TAAs) New York esophageal squamous cell carcinoma 1 (NY-ESO-1), tyrosinase, melanoma-associated antigen 3 (MAGE-A3), and transmembrane phosphatase with tensin homology (TPTE). A previous exploratory interim analysis showed that BNT111, alone or combined with immune checkpoint inhibition (CPI), has a favorable adverse event (AE) profile, gives rise to antigen-specific T-cell responses and induces durable objective responses in CPI-experienced patients with unresectable melanoma.1 Here, we present preliminary data in patients with no evidence of disease (NED) at trial inclusion in the BNT111 monotherapy subgroup.MethodsPatients with stage IIIB/C and IV pre-treated cutaneous melanoma were intravenously administered with BNT111 using a prime/repeat boost protocol. Patients were treated in seven dose escalation cohorts (7.2 to 400 µg total RNA) and three expansion cohorts to further explore dose levels of 14.4, 50 and 100 μg. In this analysis, patients receiving BNT111 monotherapy were grouped as having evidence of disease (ED) or NED, and immunogenicity, efficacy and safety were evaluated. Vaccine-induced immune responses were analyzed using an interferon-γ enzyme-linked immune absorbent spot (ELISpot) assay directly ex vivo.ResultsAs of May 24, 2021, 115 patients have received BNT111 within the Lipo MERIT trial. Of 71 patients treated with BNT111 monotherapy, 38 patients had ED and 33 patients had NED after prior therapies. Baseline characteristics were similar between the two groups. ELISpot data revealed comparable BNT111-induced T-cell responses against at least one TAA in ED vs. NED patients (14/22 [64%] and 19/28 [68%] patients with available ELISpot-evaluable samples, respectively), suggesting that BNT111 has the ability to induce T-cell immunity irrespective of the presence of a detectable tumor. As previously reported for ED patients, vaccine-induced CD4+ as well as CD8+ T-cell responses were also observed in NED patients, with a substantial fraction of de novo induced responses undetectable prior to vaccination. In NED patients, clinical efficacy was promising; median disease-free survival was 34.8 months (95% confidence interval: 7.0–not reached). The safety profile was similar in ED vs. NED patients; 38/38 (100%) and 32/33 (97%) patients experienced related treatment-emergent AEs, respectively, of which the majority were mild-to-moderate flu-like symptoms.ConclusionsImmunogenicity and safety profiles of BNT111 monotherapy were comparable in ED and NED patients. Promising signs of clinical activity were observed in NED patients.AcknowledgementsThe authors would like to acknowledge Camilla West (BioNTech SE) for medical writing support.Trial RegistrationClinicaltrials.gov: NCT02410733; EudraCT No. 2013-001646-33.ReferencesSahin U, Oehm P, Derhovanessian E, et al. An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma. Nature 2020;585(7823):107–112.Ethics ApprovalEthics &amp; Institutional Review Board approval was obtained prior to initiation of the trial (2018-13393_21-AMG federführend).

421 A first-in-human (FIH) phase I/IIa clinical trial assessing a ribonucleic acid lipoplex (RNA-LPX) encoding shared tumor antigens for immunotherapy of prostate cancer; preliminary analysis of PRO-MERIT

BackgroundPRO-MERIT is a FIH, open-label, multicenter, Phase I/IIa trial investigating a liposomal RNA vaccine (BNT112) targeting the prostate cancer tumor-associated antigens (TAAs) kallikrein-2, kallikrein-3, acid phosphatase prostate, homeobox B13 (HOXB13), and NK3 homeobox 1. BNT112 is being investigated as monotherapy and in combination with cemiplimab in patients with metastatic castration-resistant prostate cancer (mCRPC) and newly diagnosed high risk localized prostate cancer (LPC).MethodsThe trial involves dose titration in mCRPC patients (who have progressed after at least 2 but no more than 3 lines of systemic therapy) with BNT112 monotherapy (Part 1, fully recruited), followed by expansion cohorts (Part 2, recruiting) in both mCPRC and LPC with either BNT112 as monotherapy or in combination with cemiplimab. Primary trial endpoints investigate safety, tolerability, and preliminary anti-tumor activity (by Prostate Cancer Working Group 3 criteria). Secondary endpoints include determination of systemic induction or expansion of vaccine antigen-specific T cells. Vaccine-induced immune responses are analyzed ex vivo using an interferon-γ enzyme-linked immune absorbent spot (ELISpot) assay and following short-term in vitro stimulation.ResultsAs of 17 May 2021, 11 patients have received BNT112 monotherapy (9 Part 1; 2 in Part 2) and 3 patients have received BNT112 in combination with cemiplimab (at least one cycle completed). In Part 1, all 9 patients were stage IV at diagnosis and were receiving androgen deprivation therapy. Median age was 68 years. Two out of 9 patients experienced Grade 3 hypertension, leading to one dose reduction, that was initially reported as dose-limiting toxicity (DLT). All recovered within 24 h with no sequelae and the Safety Review Committee eventually concluded the events did not meet the DLT definition. Most common related adverse events (AEs) were pyrexia and hypertension. Eight serious AEs were reported in 5 patients, all unrelated to BNT112. In the 5 patients in Part 2, no additional safety signals or concerns were identified to date, either with BNT112 as monotherapy or in combination with cemiplimab. ELISpot data showed vaccine-induced immune responses were present in 7/7 ELISpot-evaluable patients. All 5 BNT112 TAAs were found to be immunogenic. Responses to each antigen were observed in at least 2 subjects. Initial responses with decreased prostate-specific antigen (PSA) levels have been observed in 2 patients in the BNT112 monotherapy arm.ConclusionsThese data suggest that BNT112 has an acceptable safety profile. Additionally, BNT112 induces robust immune and PSA responses in patients with advanced prostate cancer.AcknowledgementsThe authors would like to acknowledge Camilla West (BioNTech SE) for medical writing support.Trial RegistrationClinicaltrials.gov: NCT04382898.Ethics ApprovalEthics &amp; Institutional Review Board approvals were obtained from the respective participating countries prior to initiation of the trial.

A prospective cohort study comparing the performance of interferon gamma release assays in autoimmune skin diseases

To the Editor: Autoimmune disease patients are more likely to reactivate latent tuberculosis (TB) than the general population, and the risk increases in immunosuppressed patients.1Arenas Miras Mdel M. Hidalgo-Tenorio C. Jiménez-Gamiz P. et al.Diagnosis of latent tuberculosis in patients with systemic lupus erythematosus: T.SPOT.TB versus tuberculin skin test.Biomed Res Int. 2014; 2014: 291031PubMed Google Scholar TB screening is recommended prior to immunosuppressive therapy or clinical trial enrollment.2Jick S.S. Lieberman E.S. Rahman M.U. et al.Glucocorticoid use, other associated factors, and the risk of tuberculosis.Arthritis Rheum. 2006; 55: 9-26Crossref PubMed Scopus (308) Google Scholar,3Hamilton C.D. Tuberculosis in the cytokine era: what rheumatologists need to know.Arthritis Rheum. 2003; 48: 2085-2091Crossref PubMed Scopus (55) Google Scholar Two interferon gamma release assays (IGRAs), QuantiFERON-TB Gold Plus (QFT-Plus; Qiagen) and T-SPOT.TB test (Oxford Immunotec), demonstrate similar sensitivity and specificity in healthy populations.4QuantiFERON-TB Gold Frequently Asked Questions. Qiagen.https://www.quantiferon.com/wp-content/uploads/2017/05/PROM-10157_FAQs-Health-Professionals-Rev001v02.pdfDate accessed: January 3, 2020Google Scholar However, immunosuppressed patients are more likely to have an indeterminate or invalid IGRA due to an inadequate immune response.5Aabye M.G. Ravn P. PrayGod G. et al.The impact of HIV infection and CD4 cell count on the performance of an interferon gamma release assay in patients with pulmonary tuberculosis.PLoS One. 2009; 4: e4220Crossref PubMed Scopus (79) Google Scholar This study compares the performance of the QFT-Plus and T-SPOT.TB tests in autoimmune skin disease patients. Clinical and laboratory information were collected from the medical records at the University of Pennsylvania. IGRAs were performed according to the manufacturers' recommendations. The QFT-Plus test results were reported as positive, negative, or indeterminate. The T-Spot.TB test results were reported as positive, negative, borderline, or invalid. The QFT-Plus test indeterminate and the T-Spot.TB test invalid results were not clinically interpretable. A borderline result was designed to increase the reliability of results close to the cut-off value to reduce the incidence of false negatives or positives. Invalid T-Spot.TB test results were considered equivalent to QFT-Plus test indeterminate results. Statistical analysis was performed using the McNemar and Fisher tests. Clinical characteristics of the patients (n = 101) are described in Table I. Lymphopenia was defined as <1000 lymphocytes/μL, and 16 (15.8%) patients were diagnosed with lymphopenia. A total of 26.7% of patients did not undergo lymphocyte count testing within the 6 months. Sixteen cases (15.6%) showed indeterminate QFT-Plus test results, and 1 case (1.0%) showed an invalid T-SPOT.TB test result. One case tested positive for both IGRAs. There was a significantly higher frequency of indeterminate QFT-Plus test results compared with invalid T-SPOT.TB test results (P < .05, odds ratio 16 [2.49-670.96]). No associations between age (P = .68), immunosuppressive medication (P = .44), hydroxychloroquine (P = .77), combined antimalarial use (P = .23), lupus erythematosus (P = .49) or dermatomyositis (P = .90) diagnoses, lymphopenia (P = .72), and indeterminate/invalid results were found. Eleven patients had a previous indeterminate QuantiFERON Gold result, of whom 6 (55%) had a subsequent indeterminate QFT-Plus result upon retesting.Table IClinical characteristics and results of tuberculosis screening of 101 autoimmune skin disease patientsCharacteristicsIndeterminate/invalid (n = 17)Determinate (n = 84)P valueMedian age, y (IQR)55 (41.0-62.5)57 (44.0-62.8).68Sex, n (%)N/A Male1 (5.9)15 (17.9) Female16 (94.1)69 (82.1)Race, n (%)N/A Black2 (11.8)18 (21.4) White14 (82.3)64 (76.2) Asian1 (5.9)2 (2.4)Ethnicity, n (%)N/A Hispanic0 (0.0)4 (4.8) Non-Hispanic17 (100.0)80 (95.2)Diagnosis, n (%) Lupus7 (41.2)29 (34.5).49 Dermatomyositis9 (52.9)35 (41.7).90 Other∗Other autoimmune skin disorder diagnoses include morphea, mixed connective tissue disease, undifferentiated connective tissue disease, sarcoidosis, scleroderma, Sjogren syndrome, Sweet syndrome, eosinophilic fasciitis, erythema multiforme, interstitial granulomatous dermatitis, lichen planus pilaris, and mucous membrane pemphigoid.1 (5.9)20 (23.8)N/AHCQ use, n (%) No4 (23.5)16 (19.0).77 Yes13 (76.5)68 (81.0)Immunosuppressive medication use,†Immunosuppressive medications include methotrexate, mycophenolate, azathioprine, intravenous immunoglobulin, dapsone, and cyclosporine. n (%) No4 (23.5)37 (44.0).44 Yes13 (76.5)47 (56.0)Lymphopenia, n (%) No9 (53.0)49 (58.3).72 Yes4 (23.5)12 (14.3) N/A4 (23.5)23 (27.4)HCQ, Hydroxychloroquine; IQR, interquartile range, N/A, not available.∗ Other autoimmune skin disorder diagnoses include morphea, mixed connective tissue disease, undifferentiated connective tissue disease, sarcoidosis, scleroderma, Sjogren syndrome, Sweet syndrome, eosinophilic fasciitis, erythema multiforme, interstitial granulomatous dermatitis, lichen planus pilaris, and mucous membrane pemphigoid.† Immunosuppressive medications include methotrexate, mycophenolate, azathioprine, intravenous immunoglobulin, dapsone, and cyclosporine. Open table in a new tab HCQ, Hydroxychloroquine; IQR, interquartile range, N/A, not available. In this single-center autoimmune disease population, we demonstrated that the T-SPOT.TB test provided a definitive result significantly more often than the QFT-Plus test. One reason may be due to the differing immunological technique. The QFT-Plus test uses enzyme-linked immunosorbent assay to measure interferon gamma at the whole-blood level, whereas the TSPOT.TB test uses enzyme-linked immune absorbent spot to detect interferon gamma production at a single cell level. Additionally, the T-SPOT.TB test utilizes a cell counting technique to use fixed lymphocyte numbers prior to running the assay, which is beneficial for patients with lymphopenia. However, these findings may not be translatable to TB testing of psoriasis patients receiving biologics. Based on our findings, approximately half of patients with an indeterminate QFT-Plus will have to retest, which can be financially burdensome and delay clinical trial enrollment. Establishing the best TB screening for these patients is clinically important as inconclusive IGRA results can delay critical therapies. This study suggests that in the case of a QFT-Plus test indeterminate result, the T-SPOT.TB test should be the first-line TB-screening test in the autoimmune skin disease population. None disclosed.

Hepatitis E Infection in a Longitudinal Cohort of Hepatitis C Virus and HCV/HIV Coinfected Persons.

Hepatitis E virus (HEV) is thought to be common in the United States with increased prevalence in those with concomitant hepatitis C virus (HCV) or HCV/HIV coinfection. Little is known regarding true prevalence, incidence, and antibody seroreversion in these populations. We sought to define these rates among HCV and HCV/HIV coinfected persons in the Washington, DC area. Two longitudinal cohorts of HCV and HCV/HIV coinfected subjects from the Washington, DC area were evaluated. Multiple HEV test modalities were deployed including immunoglobulin G (IgG) and immunoglobulin M (IgM) antibody testing, evaluation of antibody avidity, HEV RNA testing, and HEV enzyme-linked immune absorbent spot (ELISPOT) analysis. A total of 379 individuals were evaluated including 196 who were HCV monoinfected and 183 HCV/HIV coinfected. Anti-HEV IgG was detected and confirmed in 18.7% of the cohort at baseline. None demonstrated anti-HEV IgM positive or HEV RNA positive results. Proportions of HEV antibody prevalence did not significantly differ between groups. Longitudinal follow-up samples were available for 226 individuals with a mean follow-up time of 24 months. Seroreversion was noted in 1.8%. One HCV/HIV infected person seroconverted to HEV IgG positivity in the followed cohort. About 40% of the positive population demonstrated high avidity suggestive of more remote exposure. Interferon gamma ELISPOT was performed in 70 subjects and false negative and false positive HEV enzyme-linked immunosorbent assay antibodies were identified. In HIV-infected persons in the United States HEV exposure and seroconversion is frequent enough that HEV should be considered in the differential diagnosis of acute hepatitis. Seroreversion may lead to underestimation of true infection risk.

Rituximab and Corticosteroid Effect on Desmoglein-Specific B Cells and Desmoglein-Specific T Follicular Helper Cells in Pemphigus

Pemphigus is an autoimmune blistering disease mediated by autoantibodies directed against desmogleins (DSGs). We recently showed that first-line treatment with rituximab (RTX) enables more patients to achieve long-lasting remission off therapy than corticosteroids alone. To understand the immunological mechanisms that mediate long-lasting clinical remission after RTX treatment, we analyzed the phenotype of DSG-specific memory B cells and DSG-specific T follicular helper cells by flow cytometry and measured antibody-secreting cells by enzyme-linked immune absorbent spot in patients treated with corticosteroids alone or RTX. This post hoc analysis of the RITUX3 trial showed that RTX induced a significant decrease of IgG-switched DSG-specific memory B cells. Accordingly, anti-DSG antibody-secreting cells were no longer detected in patients in complete remission after RTX. In contrast, corticosteroids did not modify the frequency or the phenotype of DSG-specific memory B cells, and anti-DSG antibody-secreting cells were still detected after treatment, even in patients in remission. Using peptide-HLADRB1∗0402 tetramer staining, we identified DSG-3-specific T follicular helper cells, which dramatically decreased after RTX, while remaining stable after corticosteroid treatment. Our findings suggest that long-lasting response to RTX in pemphigus relies on the decrease of DSG-specific circulating T follicular helper cells, which correlates with a sustained depletion of IgG-switched memory autoreactive B cells, leading to the disappearance of anti-DSG antibody-secreting cells.