The variable origins of persistent breathlessness after coronavirus disease 2019 (COVID-19) have hindered efforts to decipher the immunopathology of lung sequelae. Here we analyzed hundreds of cellular and molecular features in the context of discrete pulmonary phenotypes to define the systemic immune landscape of post-COVID lung disease. Cluster analysis of lung physiology measures highlighted two phenotypes of restrictive lung disease that differed according to their impaired diffusion and severity of fibrosis. Machine learning revealed marked CCR5+CD95+CD8+ T cell perturbations in milder lung disease but attenuated T cell responses hallmarked by elevated CXCL13 in more severe disease. Distinct sets of cells, mediators and autoantibodies distinguished each restrictive phenotype and differed from those of patients without substantial lung involvement. These differences were reflected in divergent T cell-based type 1 networks according to the severity of lung disease. Our findings, which provide an immunological basis for active lung injury versus advanced disease after COVID-19, might offer new targets for treatment.

Pulmonary complications arising from severe COVID-19 illness are common. However, patient variability presents a barrier to defining the immune mechanisms involved. We describe a novel method that leverages hundreds of cellular and molecular combinations within a large recovery cohort to identify immune profiles linked to discrete pulmonary phenotypes. Circulating cells and plasma mediators were monitored longitudinally for up to 2 years after acute illness using spectral flow cytometry, SARS-CoV-2 peptide stimulation assays, and multiplex bead assays (76 analytes) in a cohort of COVID-19 patients who presented for pulmonary follow-up care (n=110). Most patients were hospitalized (80%) during acute illness, and 51% received mechanical ventilation. Unsupervised machine-learning approaches (UMAP, self-organizing maps, T-REX) were used to define discrete pulmonary phenotypes and associated T-cell signatures. Analysis of multiple lung physiology measures identified 5 distinct pulmonary phenotypes that discriminated patients by clinical severity and fibrosis, and mapped trajectories of recovery versus persistence over time across these phenotypes. The most severe phenotypes were characterized by oxygen desaturation on exertion, lung restriction, and fibrosis. Related immune profiles comprised novel mixtures of complex CD4+ and CD8+ T-cell signatures that were persistently activated and marked by T-bet, PD-1 and lung-homing receptors, and increased virus-responsive T cells. Notably, CXCL13 and IFN-g discriminated the most severe pulmonary phenotype. Our novel approaches implicate dynamic pro-fibrotic, type 1-like cell networks in pulmonary complications arising after severe COVID-19. The findings deviate from current paradigms of fibrotic lung disease, and identify new mechanistic opportunities for studying lung disease in at-risk patients.

Long-haul respiratory symptoms are common following severe COVID-19 illness; however, the immune mechanisms contributing to post-acute sequelae of COVID-19 (PASC) remain enigmatic. A comprehensive 2-year longitudinal immune assessment was performed in a cohort of PASC patients receiving pulmonary follow-up care (n=110) by analyzing circulating immune cells using spectral flow cytometry, as well as assessment of virus-specific T cell frequencies, serum antibodies, and plasma mediators. Novel unsupervised machine-learning workflows were used to analyze and integrate multiple clinical and immune measures. Our approach identified discrete respiratory phenotypes according to lung physiology measures that defined the degree of lung fibrosis and its severity. Notably, severe respiratory phenotypes were characterized by immune dysregulation of both innate and adaptive cellular components, including lower ILC-3-like cells and higher activated CD4+ and CD8+ T cells in the blood. On the other hand, all respiratory phenotypes displayed a marked loss of naïve CD8+ T cells and unswitched memory B cells compared to healthy individuals. Spike- and nucleoprotein-specific CD4+ and CD8+ T cells were more abundant in severe respiratory phenotypes. Overall, T cell-derived inflammatory cytokines, along with chemokine receptors and their relationships to cognate ligands, pointed to sustained lung-homing of pathogenic effector cells in fibrotic phenotypes. Integration of high-dimensional data defined discrete cellular and molecular immune networks that provide unconventional and novel mechanistic insight into pulmonary complications post-COVID-19. UO1AI125056 R21 AI160334

Three COVID-19 vaccines have received FDA-authorization and are in use in the United States, but there is limited head-to-head data on the durability of the immune response elicited by these vaccines. Using a quantitative assay we studied binding IgG antibodies elicited by BNT162b2, mRNA-1273 or Ad26.COV2.S in an employee cohort over a span out to 10 months. Age and sex were explored as response modifiers. Of 234 subjects in the vaccine cohort, 114 received BNT162b2, 114 received mRNA-1273 and six received Ad26.COV2.S. IgG levels measured between seven to 20 days after the second vaccination were similar in recipients of BNT162b2 and mRNA-127 and were ~50-fold higher than in recipients of Ad26.COV2.S. However, by day 21 and at later time points IgG levels elicited by BNT162b2 were lower than mRNA-1273. Accordingly, the IgG decay curve was steeper for BNT162b2 than mRNA-1273. Age was a significant modifier of IgG levels in recipients of BNT162b2, but not mRNA-1273. After six months, IgG levels elicited by BNT162b2, but not mRNA-1273, were lower than IgG levels in patients who had been hospitalized with COVID-19 six months earlier. Similar findings were observed when comparing vaccine-elicited antibodies with steady-state IgG targeting seasonal human coronaviruses. Differential IgG decay could contribute to differences observed in clinical protection over time between BNT162b2 and mRNA-1273.

Some patients who survive severe COVID-19 develop persistent respiratory symptoms. Although increased activation of T-cells has been reported in severe acute disease, little is known about the long-term evolution of T-cells after SARS-CoV-2 infection in patients with long-haul symptoms. Circulating T-cells were tracked in a sample of a COVID-19 cohort (n=88) consisting of patients with persistent respiratory symptoms. Cells were obtained during severe acute COVID-19 illness and at 6 weeks and 6-11 months after hospital discharge. Cells were analyzed by high-dimensional immunophenotyping using spectral flow cytometry. Longitudinal changes in complex cell signatures were identified using the T-REX algorithm. Antibodies to SARS-CoV-2 proteins were assessed by ImmunoCAP assay. Patients with long-haul symptoms who were sampled at 6 weeks after hospital discharge had higher frequencies of activated (HLA-DR+CD38+) and tissue-homing (CCR5+) CD4+ and CD8+ T-cells compared to healthy subjects and patients with mild acute COVID-19. Higher numbers of terminally differentiated (CCR7-CD27-) CD8+ T-cells were also evident. T-REX identified multiple CD4+ and CD8+ T cell signatures that expanded or contracted by ≥95% up to 6 months after acute infection, including highly activated subtypes (CD3+6CD4+7CD45RA+7CD45RO+1CD38+1HLA-DR+4CD95+1T-BET+2TCF1+1KI-67+2 and CD3+6CD8+9CD45RA+6CD38+1HLA-DR+5CD95+2CCR5+2TCF1+1T-BET+6KI-67+2). Fluxes in T-cell signatures were detectable several months after acute infection, even in the presence of declining antibodies to SARS-CoV-2 proteins. Patients with long-haul respiratory symptoms after severe COVID-19 illness display activated T-cell signatures and marked immune perturbations, consistent with trafficking of T-cells with pathogenic potential and dysregulated homeostasis. Activated T-cells may contribute to airway inflammation long after acute illness resolves.

BACKGROUND:BNT162b2 (Pfizer/BioNTech, Comirnaty) and mRNA-1273 (Moderna, Spikevax) are messenger RNA (mRNA) vaccines that elicit antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (S-RBD) and have been approved by the US Food and Drug Administration to combat the coronavirus disease 2019 (COVID-19) pandemic. Because vaccine efficacy and antibody levels waned over time after the 2-shot primary series, the US Food and Drug Administration authorized a booster (third) dose for both mRNA vaccines to adults in the fall of 2021. OBJECTIVE:To evaluate the magnitude and durability of S-RBD immunoglobulin (Ig)G after the booster mRNA vaccine dose in comparison to the primary series. We also compared S-RBD IgG levels after BNT162b2 and mRNA-1273 boosters and explored effects of age and prior infection. METHODS:Surrounding receipt of the second and third homologous mRNA vaccine doses, adults in an employee-based cohort provided serum and completed questionnaires, including information about previous COVID-19 infection. The IgG to S-RBD was measured using an ImmunoCAP-based system. A subset of samples were assayed for IgG to SARS-CoV-2 nucleocapsid by commercial assay. RESULTS:There were 228 subjects who had samples collected between 7 and 150 days after their primary series vaccine and 117 subjects who had samples collected in the same time frame after their boost. Antibody levels from 7 to 31 days after the primary series and booster were similar, but S-RBD IgG was more durable over time after the boost, regardless of prior infection status. In addition, mRNA-1273 post-boost antibody levels exceeded BNT162b2 out to 5 months. CONCLUSION:The COVID-19 mRNA vaccine boosters increase antibody durability, suggesting enhanced long-term clinical protection from SARS-CoV-2 infection compared with the 2-shot regimen.

Background: Viral infections, especially those caused by rhinovirus, are the most common cause of asthma exacerbations. Previous studies have argued that impaired innate antiviral immunity and, as a consequence, more severe infections contribute to these exacerbations.Objective: These studies explored the innate immune response in the upper airway of volunteers with allergic rhinitis and asthma in comparison to healthy controls and interrogated how these differences corresponded to severity of infection.Methods: Volunteers with allergic rhinitis, those with asthma, and those who are healthy were inoculated with rhinovirus A16 and monitored for clinical symptoms. Tissue and nasal wash samples were evaluated for antiviral signature and viral load.Results: Both subjects with allergic rhinitis and asthma were found to have more severe cold symptoms. Subjects with asthma had worsened asthma control and increased bronchial hyperreactivity in the setting of higher fractional exhaled breath nitric oxide and blood eosinophils. These studies confirmed reduced expression of interferons and virus-specific pattern recognition receptors in both cohorts with atopy. Nevertheless, despite this defect in innate immunity, volunteers with allergic rhinitis/asthma had reduced rhinovirus concentrations in comparison to the controls.Conclusion: These results confirm that the presence of an allergic inflammatory disorder of the airway is associated with reduced innate immune responsive to rhinovirus infection. Despite this, these volunteers with allergy have reduced viral loads, arguing for the presence of a compensatory mechanism to clear the infection. Trial Registration: ClinicalTrials.gov Identifier: NCT02910401. (c) 2022 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Infection with rhinovirus (RV) is a major risk factor for disease exacerbations in patients with allergic asthma. This study analysed a broad set of cytokines in the noses of children and adults with asthma during RV infection in order to identify immunophenotypes that may link to virus‐induced episodes.

Background Rhinovirus frequently causes asthma exacerbations among children and young adults who are allergic. The interaction between allergen and rhinovirus-induced symptoms and inflammation over time is unclear. Objective Our aim was to compare the response to an experimental inoculation with rhinovirus-16 in allergic asthmatics with the response in healthy controls and to evaluate the effects of administrating omalizumab before and during the infection. Methods Two clinical trials were run in parallel. In one of these trials, the response to an experimental inoculation with rhinovirus-16 among asthmatics with high levels of total IgE was compared to the response in healthy controls. The other trial compared the effects of administering omalizumab versus placebo to asthmatics in a randomized, double-blind placebo-controlled investigation. The primary outcome for both trials compared lower respiratory tract symptoms (LRTSs) between study groups over the first 4 days of infection. Results Frequent comparisons of symptoms, lung function, and blood eosinophil counts revealed differences that were more pronounced among allergic asthmatics than among controls by days 2 and 3 after virus inoculation. Additionally, an augmentation of upper respiratory tract symptom scores and LRTS scores occurred among the atopic asthmatics versus the controls during the resolution of symptoms (P < .01 for upper respiratory symptom tract scores and P < .001 for LRTS scores). The beneficial effects of administering omalizumab on reducing LRTSs and improving lung function were strongest over the first 4 days. Conclusions LRTSs and blood eosinophil counts were augmented and lung function was reduced among allergic asthmatics early after rhinovirus inoculation but increased late in the infection during symptom resolution. The effect of administering omalizumab on the response to rhinovirus was most pronounced during the early/innate phase of the infection.

Asthma exacerbations triggered by rhinovirus (RV) have been ascribed to reduced innate immunity. However, previous studies have not compellingly demonstrated higher viral loads in allergic or asthmatic subjects and therapeutic interventions to enhance innate immune responses have not proven effective in preventing exacerbations. We therefore investigated alternative mechanisms that could explain restriction of RV infection. RV challenges were performed in healthy control (HC), allergic rhinitis (AR), and asthma cohorts and viral load post infection was quantified. Nasal lavage fluid and nasal scrapingscollected on days 1,2,3,4, and 7 post-infectionwere assayed for innate immune and type 2 inflammation transcripts and proteosomes. Nasal biopsies were obtained on day 4 post-infection. AR and asthma cohorts demonstrated worse and more protracted symptoms compared to HC. Despite this, viral loads were highest in the HC subjects. AR and asthma cohorts demonstrated reduced innate immunity including decreased expression of interferon-aand TLR3 (by qPCR and immunohistochemistry). AR and asthma subjects demonstrated enhanced eosinophilic inflammation, including nasal lavage eosinophil-derived neurotoxin (EDN) and tissue EDN and major basic protein. Eosinophils and eosinophil-derived mediators (EDN) were shown to mediate potent anti-RV efficacy. AR and asthma are characterized by reduced innate immunity and worsened symptoms after RV inoculation. Despite this, these subjects had lower viral loads. Recruitment of eosinophils in allergic and asthmatic subjects may restrict RV and compensate for the reduced innate immunity. However, this anti-viral efficacy of eosinophils may be accomplished at the cost of worsened symptoms driven by increased eosinophilic and type 2 inflammation.

Background: Allergic asthmatic subjects are uniquely susceptible to acute wheezing episodes provoked by rhinovirus. However, the underlying immune mechanisms and interaction between rhinovirus and allergy remain enigmatic, and current paradigms are controversial. Objective: We sought to perform a comprehensive analysis of type 1 and type 2 innate and adaptive responses in allergic asthmatic subjects infected with rhinovirus. Methods: Circulating virus-specific T(H)1 cells and allergenspecific T(H)2 cells were precisely monitored before and after rhinovirus challenge in allergic asthmatic subjects (total IgE, 133-4692 IU/mL; n = 28) and healthy nonallergic controls (n = 12) using peptide/MHCII tetramers. T cells were sampled for up to 11 weeks to capture steady-state and postinfection phases. T-cell responses were analyzed in parallel with 18 cytokines in the nose, upper and lower airway symptoms, and lung function. The influence of in vivo IgE blockade was also examined. Results: In uninfected asthmatic subjects, higher numbers of circulating virus-specific PD-1(+) T(H)1 cells, but not allergenspecific T(H)2 cells, were linked to worse lung function. Rhinovirus infection induced an amplified antiviral T(H)1 response in asthmatic subjects versus controls, with synchronized allergen-specific T(H)2 expansion, and production of type 1 and 2 cytokines in the nose. In contrast, T(H)2 responses were absent in infected asthmatic subjects who had normal lung function, and in those receiving anti-IgE. Across all subjects, early induction of a minimal set of nasal cytokines that discriminated high responders (G-CSF, IFN-gamma, TNF-alpha) correlated with both egress of circulating virus-specific T(H)1 cells and worse symptoms. Conclusions: Rhinovirus induces robust T(H)1 responses in allergic asthmatic subjects that may promote disease, even after the infection resolves.

The percentage of total IgE specific for an allergen in serum and on basophils has been shown to correlate. We evaluated whether the ratio of specific IgE for peanut/total IgE in serum differs among children who pass or fail a food challenge. Twenty four children ≥ 3 yrs of age underwent a peanut food challenge. Previous reactions to peanut, prick skin test (PST) results, and serum assessments of total IgE, peanut specific IgE, and IgE to peanut components were evaluated. Ten children were sensitized to Arah 1 and/or 2. Five FAILED their challenge (reacting to ≤ 1.8 grams peanut protein); median age and PST size = 4 yrs and 5mm. Five PASSED their challenge; median age and PST size = 5 yrs and 7 mm. Four in each group experienced a previous reaction to peanut. Specific IgE to peanut was 3.4% of the total IgE among those who FAILED their challenge compared to 0.8% among those who PASSED (p = 0.23). By comparison, specific IgE to peanut was 0.3% of total IgE among 14 children who PASSED the challenge and lacked IgE ab to Arah 1 or 2 (p = 0.01 compared to the 5 sensitized to Arah 1 or 2 who FAILED the challenge; p = 0.18 compared to those who PASSED). The percentage of total IgE specific for peanut was higher among children who failed their challenge and may serve to judge the safety of a peanut food challenge.

Rhinovirus (RV) infections frequently cause asthma exacerbations in children and young adults. RV infections are also known to stimulate recruitment of neutrophils into the airways. We speculate that neutrophil mediators will be increased in nasal washes (NW’s) from asthmatics compared to non-asthmatic controls during an experimental infection with RV-16. Sixteen subjects (ages 19-33) were inoculated with RV-16 (dose=300 TCID50). They included 9 allergic-asthmatics (total IgE levels 596-1989 IU/mL), and 7 controls (total IgE levels 5-42 IU/mL). Neutrophil mediators, neutrophil elastase (NE) and myeloperoxidase (MPO), were measured by ELISA in NW’s obtained before and during (Days 1, 2, 3, 4, 7, 14, 21) the infection. The results were compared to symptom scores. Both NE and MPO levels peaked in NW’s on Day 3 of the infection, paralleling the development of peak cold symptoms in both asthmatics and controls. Compared to baseline values before inoculation, mediator values by Day 3 increased 7 and 5-fold for NE and MPO among asthmatics, respectively, and 5 and 3-fold among the controls. Cumulative values (summed over days 1-3 following RV inoculation) trended higher in asthmatics compared to controls (e.g., geometric means for NE=3775ng/mL and 2167ng/mL, respectively, p=0.45; MPO=1375ng/mL and 917.3ng/mL, p=0.66), but this trend was not apparent during resolution of the infection (days 14 and 21). During the early (innate) phase of the infection, a consistent trend in higher levels of neutrophil mediators was observed in nasal washes from asthmatic subjects compared to controls. This increase was no longer seen during resolution of the infection.

Background Rhinovirus (HRV) is associated with the large majority of virus-induced asthma exacerbations in children and young adults, but the mechanisms remain poorly defined. Methods Asthmatics and non-asthmatic controls were inoculated with HRV-A16, and nasal epithelial samples were obtained 7 days before, 36 hours after, and 7 days after viral inoculation. RNA was extracted and subjected to RNA-seq analysis. Results At baseline, 57 genes were differentially expressed between asthmatics and controls, and the asthmatics had decreased expression of viral replication inhibitors and increased expression of genes involved in inflammation. At 36 hours (before the emergence of peak symptoms), 1329 genes were significantly altered from baseline in the asthmatics compared to 62 genes in the controls. At this time point, asthmatics lacked an increase in IL-10 signaling observed in the controls. At 7 days following HRV inoculation, 222 genes were significantly dysregulated in the asthmatics, whereas only 4 genes were dysregulated among controls. At this time point, the controls but not asthmatics demonstrated upregulation of SPINK5. Conclusions As judged by the magnitude and persistence of dysregulated genes, asthmatics have a substantially different host response to HRV-A16 infection compared with non-asthmatic controls. Gene expression differences illuminate biologically plausible mechanisms that contribute to a better understanding of the pathogenesis of HRV-induced asthma exacerbations.

Rhinovirus (RV) infections frequently cause asthma exacerbations in children and young adults. Mechanisms, including the capacity of RV to stimulate Th2 related responses, remain unclear. Twelve subjects (ages 19-33) were inoculated with RV 16 (dose = 300 TCID50). They included 7 allergic-asthmatics (AA; total IgE levels 596-1989 IU/mL), and 5 non-atopic controls without asthma (total IgE levels 5 to 42 IU/mL). Eosinophil mediators (ECP [Phadia AB] and EDN [MBL International Corporation]) were measured by ELISA in nasal washes (NW's) obtained before and during the infection. The results were analyzed in relation to symptoms. Both ECP and EDN peaked by day 3, paralleling cold symptoms over the first 4 days of the infection. Cumulative values derived from morning NWs during the first four days were significantly higher among AA subjects than controls (ECP: GM = 416 ng/mL and 16.8 ng/mL, respectively, p < 0.05; EDN: GM = 1320 ng/mL and 260.6 ng/mL, respectively, p < 0.05). Compared to baseline values (determined before inoculation), mediator values in NW's increased 20-fold for ECP and 16.6-fold for EDN by day 3 in the AA subjects. By comparison, ECP and EDN levels increased 2.6-fold and 4.7-fold, respectively, among controls. The increase in eosinophil mediators (ECP and EDN) in nasal washes after RV inoculation was significantly greater in the allergic asthmatics than controls, but the results indicate that RV may also have the capacity to stimulate a Th2 related eosinophil response in the non-allergic, non-asthmatic host.