Investigating viral and autoimmune T cell responses associated with post-acute sequelae of COVID-19.

Post-acute sequelae of COVID-19 (PASC), or Long COVID, is a chronic condition following acute SARS-CoV-2 infection. Symptoms include exertion fatigue, respiratory issues, myalgia, and neurological manifestations such as 'brain fog,' posing concern for their debilitating nature and potential role in other neurological disorders. However, the underlying potential pathogenic mechanisms of the neurological complications of PASC is largely unknown. Herein, we investigated differences in antigen-specific T cell responses from the peripheral blood towards SARS-CoV-2, latent viruses, or neuronal antigens in 14 PASC individuals with neurological manifestations (PASC-N) versus 22 individuals fully recovered from COVID-19. We employed Activation Induced Marker (AIM), ICS and FluoroSpot assays to determine the specificity and magnitude of CD4+ and CD8+ T cell responses towards SARS-CoV-2 (Spike and rest of proteome), latent viruses (CMV, EBV), and several neuronal antigens. Overall, we observed similar antigen-specific T cell frequencies and cytokine effector T cell responses between PASC donors compared to recovered controls for all antigens tested (viral or autoantigen) in both CD4+ and CD8+ T cell compartments. Our findings suggest that PASC-N does not appear to be associated with changes in antigen-specific T cell responses towards a subset of disease-relevant targets, but more studies in a larger cohort are needed to confirm these unaltered responses.

Immunological memory to common cold coronaviruses assessed longitudinally over a three-year period pre-COVID19 pandemic.

The immune memory to common cold coronaviruses (CCCs) influences SARS-CoV-2 infection outcome, and understanding its effect is crucial for pan-coronavirus vaccine development. We performed a longitudinal analysis of pre-COVID19-pandemic samples from 2016-2019 in young adults and assessed CCC-specific CD4+ T cell and antibody responses. Notably, CCC responses were commonly detected with comparable frequencies as with other common antigens and were sustained over time. CCC-specific CD4+ T cell responses were associated with low HLA-DR+CD38+ signals, and their magnitude did not correlate with yearly CCC infection prevalence. Similarly, CCC-specific and spike RBD-specific IgG responses were stable in time. Finally, high CCC-specific CD4+ T cell reactivity, but not antibody titers, was associated with pre-existing SARS-CoV-2 immunity. These results provide a valuable reference for understanding the immune response to endemic coronaviruses and suggest that steady and sustained CCC responses are likely from a stable pool of memory CD4+ T cells due to repeated earlier exposures and possibly occasional reinfections.

Transcriptomics of Acute DENV-Specific CD8+ T Cells Does Not Support Qualitative Differences as Drivers of Disease Severity.

While several lines of evidence suggest a protective role of T cells against disease associated with Dengue virus (DENV) infection, their potential contribution to immunopathology in the acute phase of DENV infection remains controversial, and it has been hypothesized that the more severe form of the disease (dengue hemorrhagic fever, DHF) is associated with altered T cell responses. To address this question, we determined the transcriptomic profiles of DENV-specific CD8+ T cells in a cohort of 40 hospitalized dengue patients with either a milder form of the disease (dengue fever, DF) or a more severe disease form (dengue hemorrhagic fever, DHF). We found multiple transcriptomic signatures, one associated with DENV-specific interferon-gamma responding cells and two other gene signatures, one specifically associated with the acute phase and the other with the early convalescent phase. Additionally, we found no differences in quantity and quality of DENV-specific CD8+ T cells based on disease severity. Taken together with previous findings that did not detect altered DENV-specific CD4 T cell responses, the current analysis argues against alteration in DENV-specific T cell responses as being a correlate of immunopathology.

Immunological memory to Common Cold Coronaviruses assessed longitudinally over a three-year period.

Understanding immune memory to Common Cold Coronaviruses (CCCs) is relevant for assessing its potential impact on the outcomes of SARS-CoV-2 infection, and for the prospects of pan-corona vaccines development. We performed a longitudinal analysis, of pre-pandemic samples collected from 2016-2019. CD4+ T cells and antibody responses specific for CCC and to other respiratory viruses, and chronic or ubiquitous pathogens were assessed. CCC-specific memory CD4+ T cells were detected in most subjects, and their frequencies were comparable to those for other common antigens. Notably, responses to CCC and other antigens such as influenza and Tetanus Toxoid (TT) were sustained over time. CCC-specific CD4+ T cell responses were also associated with low numbers of HLA-DR+CD38+ cells and their magnitude did not correlate with yearly changes in the prevalence of CCC infections. Similarly, spike RBD-specific IgG responses for CCC were stable throughout the sampling period. Finally, high CD4+ T cell reactivity to CCC, but not antibody responses, was associated with high pre-existing SARS-CoV-2 immunity. Overall, these results suggest that the steady and sustained CCC responses observed in the study cohort are likely due to a relatively stable pool of CCC-specific memory CD4+ T cells instead of fast decaying responses and frequent reinfections.

SARS-CoV-2 vaccination induces immunological T cell memory able to cross-recognize variants from Alpha to Omicron.

We address whether T cell responses induced by different vaccine platforms (mRNA-1273, BNT162b2, Ad26.COV2.S, and NVX-CoV2373) cross-recognize early SARS-CoV-2 variants. T cell responses to early variants were preserved across vaccine platforms. By contrast, significant overall decreases were observed for memory B cells and neutralizing antibodies. In subjects ∼6 months post-vaccination, 90% (CD4+) and 87% (CD8+) of memory T cell responses were preserved against variants on average by AIM assay, and 84% (CD4+) and 85% (CD8+) preserved against Omicron. Omicron RBD memory B cell recognition was substantially reduced to 42% compared with other variants. T cell epitope repertoire analysis revealed a median of 11 and 10 spike epitopes recognized by CD4+ and CD8+ T cells, with average preservation > 80% for Omicron. Functional preservation of the majority of T cell responses may play an important role as a second-level defense against diverse variants.

Development of a T cell-based immunodiagnostic system to effectively distinguish SARS-CoV-2 infection and COVID-19 vaccination status.

Both SARS-CoV-2 infections and COVID-19 vaccines elicit memory T cell responses. Here, we report the development of 2 pools of experimentally defined SARS-CoV-2 T cell epitopes that, in combination with spike, were used to discriminate 4 groups of subjects with different SARS-CoV-2 infection and COVID-19 vaccine status. The overall T cell-based classification accuracy was 89.2% and 88.5% in the experimental and validation cohorts. This scheme was applicable to different mRNA vaccines and different lengths of time post infection/post vaccination and yielded increased accuracy when compared to serological readouts. T cell responses from breakthrough infections were also studied and effectively segregated from vaccine responses, with a combined performance of 86.6% across all 239 subjects from the 5 groups. We anticipate that a T cell-based immunodiagnostic scheme to classify subjects based on their vaccination and natural infection history will be an important tool for longitudinal monitoring of vaccinations and for establishing SARS-CoV-2 correlates of protection.

A Population of CD4+CD8+ Double-Positive T Cells Associated with Risk of Plasma Leakage in Dengue Viral Infection.

According to the WHO 2009 classification, dengue with warning signs is at the risk of developing severe form of dengue disease. One of the most important warning signs is plasma leakage, which can be a serious complication associated with higher morbidity and mortality. We report that the frequency of CD4+CD8+ double-positive (DP) T cells is significantly increased in patients at risk of developing plasma leakage. Transcriptomic analysis demonstrated that CD4+CD8+ DP cells were distinct from CD4+ Single Positive (SP) T cells but co-clustered with CD8+ SP cells, indicating a largely similar transcriptional profile. Twenty significant differentially expressed (DE) genes were identified between CD4+CD8+ DP and CD8+ SP cells. These genes encode OX40 and CCR4 proteins as well as other molecules associated with cell signaling on the cell surface (NT5E, MXRA8, and PTPRK). While comparing the profile of gene expression in CD4+CD8+ DP cells from patients with and without warning signs of plasma leakage, similar expression profile was observed, implying a role of CD4+CD8+ DP cells in plasma leakage through a quantitative increase rather than functional alteration. This study provided novel insight into the host immune response during the acute febrile phase of DENV infection and the role of CD4+CD8+ DP T cells in the pathogenesis of plasma leakage.

Ex vivo assays show human gamma-delta T cells specific for common allergens are Th1-polarized in allergic donors.

Gamma-delta (γδ) T cells contribute to the pathology of many immune-related diseases; however, no ex vivo assays to study their activities are currently available. Here, we established a methodology to characterize human allergen-reactive γδ T cells in peripheral blood using an activation-induced marker assay targeting upregulated 4-1BB and CD69 expression. Broad and reproducible ex vivo allergen-reactive γδ T cell responses were detected in donors sensitized to mouse, cockroach, house dust mite, and timothy grass, but the response did not differ from that in non-allergic participants. The reactivity to 4 different allergen extracts was readily detected in 54.2%-100% of allergic subjects in a donor- and allergen-specific pattern and was abrogated by T cell receptor (TCR) blocking. Analysis of CD40L upregulation and intracellular cytokine staining revealed a T helper type 1 (Th1)-polarized response against mouse and cockroach extract stimulation. These results support the existence of allergen-reactive γδ T cells and their potential use in rebalancing dysregulated Th2 responses in allergic diseases.

Impact of SARS-CoV-2 variants on the total CD4+ and CD8+ T cell reactivity in infected or vaccinated individuals.

The emergence of SARS-CoV-2 variants with evidence of antibody escape highlight the importance of addressing whether the total CD4+ and CD8+ T cell recognition is also affected. Here, we compare SARS-CoV-2-specific CD4+ and CD8+ T cells against the B.1.1.7, B.1.351, P.1, and CAL.20C lineages in COVID-19 convalescents and in recipients of the Moderna (mRNA-1273) or Pfizer/BioNTech (BNT162b2) COVID-19 vaccines. The total reactivity against SARS-CoV-2 variants is similar in terms of magnitude and frequency of response, with decreases in the 10%-22% range observed in some assay/VOC combinations. A total of 7% and 3% of previously identified CD4+ and CD8+ T cell epitopes, respectively, are affected by mutations in the various VOCs. Thus, the SARS-CoV-2 variants analyzed here do not significantly disrupt the total SARS-CoV-2 T cell reactivity; however, the decreases observed highlight the importance for active monitoring of T cell reactivity in the context of SARS-CoV-2 evolution.

Balanced Cellular and Humoral Immune Responses Targeting Multiple Antigens in Adults Receiving a Quadrivalent Inactivated Influenza Vaccine.

The role of T cell immunity has been acknowledged in recent vaccine development and evaluation. We tested the humoral and cellular immune responses to Flucelvax®, a quadrivalent inactivated seasonal influenza vaccine containing two influenza A (H1N1 Singapore/GP1908/2015 IVR-180 and H3N2 North Carolina/04/2016) and two influenza B (Iowa/06/2017 and Singapore/INFTT-16-0610/2016) virus strains, using peripheral blood mononuclear cells stimulated by pools of peptides overlapping all the individual influenza viral protein components. Baseline reactivity was detected against all four strains both at the level of CD4 and CD8 responses and targeting different proteins. CD4 T cell reactivity was mostly directed to HA/NA proteins in influenza B strains, and NP/M1/M2/NS1/NEP proteins in the case of the Influenza A strains. CD8 responses to both influenza A and B viruses preferentially targeted the more conserved core viral proteins. Following vaccination, both CD4 and CD8 responses against the various influenza antigens were increased in day 15 to day 91 post vaccination period, and maintained a Th1 polarized profile. Importantly, no vaccine interference was detected, with the increased responses balanced across all four included viral strains for both CD4 and CD8 T cells, and targeting HA and multiple additional viral antigens.

B cells modulate mouse allergen-specific T cells in nonallergic laboratory animal-care workers.

Understanding the mechanisms of allergen-specific immune modulation in nonallergic individuals is key to recapitulate immune tolerance and to develop novel allergy treatments. Herein, we characterized mouse-specific T cell responses in nonallergic laboratory animal-care workers before and after reexposure to mice. PBMCs were collected and stimulated with developed peptide pools identified from high-molecular-weight fractions of mouse allergen extracts. Sizable CD4 T cell responses were noted and were temporarily decreased in most subjects upon reexposure, with the magnitude of decrease positively correlated with time of reexposure but not the duration of the break. Interestingly, the suppression was specific to mouse allergens without affecting responses of bystander antigens. Further, PBMC fractioning studies illustrated that the modulation is unlikely from T cells, while B cell depletion and exchange reversed the suppression of responses, suggesting that B cells may be the key modulators. Increased levels of regulatory cytokines (IL-10 and TGF-ß1) in the cell culture supernatant and plasma mouse-specific IgG4 were also observed after reexposure, consistent with B cell-mediated modulation mechanisms. Overall, these results suggest that nonallergic status is achieved by an active, time-related, allergen-specific, B cell-dependent regulatory process upon reexposure, the mechanisms of which should be detailed by further molecular studies.

Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases.

T cells are involved in control of SARS-CoV-2 infection. To establish the patterns of immunodominance of different SARS-CoV-2 antigens and precisely measure virus-specific CD4+ and CD8+ T cells, we study epitope-specific T cell responses of 99 convalescent coronavirus disease 2019 (COVID-19) cases. The SARS-CoV-2 proteome is probed using 1,925 peptides spanning the entire genome, ensuring an unbiased coverage of human leukocyte antigen (HLA) alleles for class II responses. For HLA class I, we study an additional 5,600 predicted binding epitopes for 28 prominent HLA class I alleles, accounting for wide global coverage. We identify several hundred HLA-restricted SARS-CoV-2-derived epitopes. Distinct patterns of immunodominance are observed, which differ for CD4+ T cells, CD8+ T cells, and antibodies. The class I and class II epitopes are combined into epitope megapools to facilitate identification and quantification of SARS-CoV-2-specific CD4+ and CD8+ T cells.

Differential T cell reactivity to seasonal coronaviruses and SARS-CoV-2 in community and health care workers.

Herein we measured CD4+ T cell responses against common cold corona (CCC) viruses and SARS-CoV-2 in high-risk health care workers (HCW) and community controls. We observed higher levels of CCC reactive T cells in SARS-CoV-2 seronegative HCW compared to community donors, consistent with potential higher occupational exposure of HCW to CCC. We further show that SARS-CoV-2 reactivity of seronegative HCW was higher than community controls and correlation between CCC and SARS-CoV-2 responses is consistent with cross-reactivity and not associated with recent in vivo activation. Surprisingly, CCC reactivity was decreased in SARS-CoV-2 infected HCW, suggesting that exposure to SARS-CoV-2 might interfere with CCC responses, either directly or indirectly. This result was unexpected, but consistently detected in independent cohorts derived from Miami and San Diego.

Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection.

Understanding immune memory to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for improving diagnostics and vaccines and for assessing the likely future course of the COVID-19 pandemic. We analyzed multiple compartments of circulating immune memory to SARS-CoV-2 in 254 samples from 188 COVID-19 cases, including 43 samples at ≥6 months after infection. Immunoglobulin G (IgG) to the spike protein was relatively stable over 6+ months. Spike-specific memory B cells were more abundant at 6 months than at 1 month after symptom onset. SARS-CoV-2-specific CD4+ T cells and CD8+ T cells declined with a half-life of 3 to 5 months. By studying antibody, memory B cell, CD4+ T cell, and CD8+ T cell memory to SARS-CoV-2 in an integrated manner, we observed that each component of SARS-CoV-2 immune memory exhibited distinct kinetics.

Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases.

T cells are involved in control of SARS-CoV-2 infection. To establish the patterns of immunodominance of different SARS-CoV-2 antigens, and precisely measure virus-specific CD4 + and CD8 + T cells, we studied epitope-specific T cell responses of approximately 100 convalescent COVID-19 cases. The SARS-CoV-2 proteome was probed using 1,925 peptides spanning the entire genome, ensuring an unbiased coverage of HLA alleles for class II responses. For HLA class I, we studied an additional 5,600 predicted binding epitopes for 28 prominent HLA class I alleles, accounting for wide global coverage. We identified several hundred HLA-restricted SARS-CoV-2-derived epitopes. Distinct patterns of immunodominance were observed, which differed for CD4 + T cells, CD8 + T cells, and antibodies. The class I and class II epitopes were combined into new epitope megapools to facilitate identification and quantification of SARS-CoV-2-specific CD4 + and CD8 + T cells.

Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed humans.

Many unknowns exist about human immune responses to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. SARS-CoV-2-reactive CD4+ T cells have been reported in unexposed individuals, suggesting preexisting cross-reactive T cell memory in 20 to 50% of people. However, the source of those T cells has been speculative. Using human blood samples derived before the SARS-CoV-2 virus was discovered in 2019, we mapped 142 T cell epitopes across the SARS-CoV-2 genome to facilitate precise interrogation of the SARS-CoV-2-specific CD4+ T cell repertoire. We demonstrate a range of preexisting memory CD4+ T cells that are cross-reactive with comparable affinity to SARS-CoV-2 and the common cold coronaviruses human coronavirus (HCoV)-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1. Thus, variegated T cell memory to coronaviruses that cause the common cold may underlie at least some of the extensive heterogeneity observed in coronavirus disease 2019 (COVID-19) disease.

Immunological memory to SARS-CoV-2 assessed for up to eight months after infection.

Understanding immune memory to SARS-CoV-2 is critical for improving diagnostics and vaccines, and for assessing the likely future course of the COVID-19 pandemic. We analyzed multiple compartments of circulating immune memory to SARS-CoV-2 in 254 samples from 188 COVID-19 cases, including 43 samples at ≥ 6 months post-infection. IgG to the Spike protein was relatively stable over 6+ months. Spike-specific memory B cells were more abundant at 6 months than at 1 month post symptom onset. SARS-CoV-2-specific CD4 + T cells and CD8 + T cells declined with a half-life of 3-5 months. By studying antibody, memory B cell, CD4 + T cell, and CD8 + T cell memory to SARS-CoV-2 in an integrated manner, we observed that each component of SARS-CoV-2 immune memory exhibited distinct kinetics.

Comparison of therapeutic interventions for recurrent pregnancy loss in association with antiphospholipid syndrome: A systematic review and network meta-analysis.

Antiphospholipid syndrome (APS) is one of the treatable causes for pregnant women with recurrent pregnancy loss (RPL). This review compares the efficacy of a few treatment interventions (low-dose aspirin (LDA), aspirin plus low molecular weight heparin (LMWH), or unfractionated heparin (UFH)) in preventing complications during pregnancy and miscarriages for women with RPL and APS, and the potential differences in therapeutic effects of UFH and LMWH when combined with aspirin. We searched randomized controlled trials (RCTs) in MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials and performed a systematic review and a Bayesian network meta-analysis (NMA). Finally, we found aspirin alone had a lower live birth rate compared to LMWH plus aspirin (OR = 0.37; 95% CrI, 0.17, 0.71), and UFH plus aspirin showed a higher live birth rate than aspirin alone (OR = 2.63; 95% CrI, 1.04, 5.39) in NMA, treating with UFH plus aspirin or LMWH plus aspirin did not have difference on live birth. Furthermore, LDA alone resulted in a lower birthweight compared to heparin plus aspirin, while higher birthweight was found when compared UFH plus aspirin to LMWH plus aspirin (MD = 895.40; 95% CrI, 817.40, 988.57) in NMA. Additionally, in women with RPL and APS and without a prior thrombosis, heparin plus aspirin improved birthweight but could not promote live birth rate compared to aspirin alone. In conclusion, heparin plus aspirin is recommended for women with RPL and APS. Notably UFH plus aspirin demonstrates the most significant therapeutic efficacy among these interventions in improving birthweight.

Comparison of the clinical performance of i-gel, LMA Supreme and LMA ProSeal in elective surgery.

INTRODUCTION: The LMA Supreme™, i-gel® and LMA ProSeal™ are second-generation supraglottic airway devices. We tested the hypothesis that these devices differ in performance when used for spontaneous ventilation during anaesthesia. METHODS: 150 patients who underwent general anaesthesia for elective surgery were randomly allocated into three groups. Data was collected on oropharyngeal leak pressures, ease and duration of device insertion, ease of gastric tube insertion, and airway safety. RESULTS: Leak pressure, our primary outcome measure, was found to be higher for the i-gel than the Supreme and ProSeal (mean ± standard error of the mean: 27.31 ± 0.92 cmH2O, 23.60 ± 0.70 cmH2O and 24.44 ± 0.70 cmH2O, respectively; p = 0.003). Devices were inserted on the first attempt for 90%, 82% and 72% of patients in the i-gel, Supreme and ProSeal groups, respectively (p = 0.105); mean device placement times were 23.58 seconds, 25.10 seconds and 26.34 seconds, respectively (p = 0.477). Gastric tubes were inserted on the first attempt in 100% of patients in the Supreme group, and 94% of patients in the i-gel and ProSeal groups (p = 0.100). There was blood staining on removal in 9 (18%) patients in each of the Supreme and ProSeal groups, with none in the i-gel group (p = 0.007). The incidence of postoperative sore throat, dysphagia and hoarseness was lowest for the i-gel. CONCLUSION: The three devices were comparable in terms of ease and duration of placement, but the i-gel had higher initial oropharyngeal leak pressure and lower airway morbidity compared with the ProSeal and Supreme.

Polymorphisms of anti-Müllerian hormone signaling pathway in healthy Singapore women: population differences, endocrine effects and reproductive outcomes.

In order to study the association of genetic polymorphisms of anti-Müllerian hormone (AMH) signaling pathway with endocrine changes and pregnancy outcomes, a total of 213 women of reproductive ages were recruited according to our inclusion and exclusion criteria between November 2011 and September 2014 in Singapore. Genotyping studies were performed using a minor groove binder primer/probe Taqman assay. The allele frequencies of the AMH Ile(49)Ser and AMHR2 -482A > G polymorphisms were analyzed in relation to female reproductive hormone levels, ovarian parameters, menstrual cycle lengths and pregnancy outcomes. AMH Ser allele frequency and AMHR2 G allele frequency of our Singapore population were compared with those of other populations reported in HapMap. The genotype distributions and allele frequencies for the AMH Ile(49)Ser and AMHR2 -482A > G polymorphisms were not associated with estradiol (E2) levels, ovarian parameters, menstrual cycle length, or pregnancy outcomes in our cohort. Our findings suggest that genetic variants in the AMH signal transduction pathway have population differences but do not appear to have significant effects on ovarian, endocrine, metabolic parameters and reproductive outcomes.