Validating a Proteomic Signature of Severe COVID-19.

COVID-19 is a heterogenous disease. Biomarker-based approaches may identify patients at risk for severe disease, who may be more likely to benefit from specific therapies. Our objective was to identify and validate a plasma protein signature for severe COVID-19. DESIGN: Prospective observational cohort study. SETTING: Two hospitals in the United States. PATIENTS: One hundred sixty-seven hospitalized adults with COVID-19. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: We measured 713 plasma proteins in 167 hospitalized patients with COVID-19 using a high-throughput platform. We classified patients as nonsevere versus severe COVID-19, defined as the need for high-flow nasal cannula, mechanical ventilation, extracorporeal membrane oxygenation, or death, at study entry and in 7-day intervals thereafter. We compared proteins measured at baseline between these two groups by logistic regression adjusting for age, sex, symptom duration, and comorbidities. We used lead proteins from dysregulated pathways as inputs for elastic net logistic regression to identify a parsimonious signature of severe disease and validated this signature in an external COVID-19 dataset. We tested whether the association between corticosteroid use and mortality varied by protein signature. One hundred ninety-four proteins were associated with severe COVID-19 at the time of hospital admission. Pathway analysis identified multiple pathways associated with inflammatory response and tissue repair programs. Elastic net logistic regression yielded a 14-protein signature that discriminated 90-day mortality in an external cohort with an area under the receiver-operator characteristic curve of 0.92 (95% CI, 0.88-0.95). Classifying patients based on the predicted risk from the signature identified a heterogeneous response to treatment with corticosteroids (p = 0.006). CONCLUSIONS: Inpatients with COVID-19 express heterogeneous patterns of plasma proteins. We propose a 14-protein signature of disease severity that may have value in developing precision medicine approaches for COVID-19 pneumonia.

Modulation of the immune response to SARS-CoV-2 vaccination by NSAIDs.

Evidence is scarce to guide the use of nonsteroidal anti-inflammatory drugs (NSAIDs) to mitigate SARS-CoV-2 vaccine related adverse effects, given the possibility of blunting the desired immune response. In this pilot study, we deeply phenotyped a small number of volunteers who did or did not take NSAIDs concomitant with SARS-CoV-2 immunizations to seek initial information on the immune response. A SARS-CoV-2 vaccine specific RBD-IgG antibody response and efficacy in the evoked neutralization titers were evident irrespective of concomitant NSAID consumption. Given the sample size, only a large and consistent signal of immunomodulation would have been detectable, and this was not apparent. However, the information gathered may inform the design of a definitive clinical trial. Here, we report a series of divergent omics signals that invite additional hypotheses testing. Significance Statement A SARS-CoV-2 vaccine specific immune response was evident irrespective of concomitant NSAID consumption in a clinical pilot study of small sample size.

An extended SARS-CoV-2 mRNA vaccine prime-boost interval enhances B cell immunity with limited impact on T cells.

Spacing the first two doses of SARS-CoV-2 mRNA vaccines beyond 3-4 weeks raised initial concerns about vaccine efficacy. While studies have since shown that long-interval regimens induce robust antibody responses, their impact on B and T cell immunity is poorly known. Here, we compare SARS-CoV-2 naive donors B and T cell responses to two mRNA vaccine doses administered 3-4 versus 16 weeks apart. After boost, the longer interval results in a higher magnitude and a more mature phenotype of RBD-specific B cells. While the two geographically distinct cohorts present quantitative and qualitative differences in T cell responses at baseline and after priming, the second dose led to convergent features with overall similar magnitude, phenotype, and function of CD4+ and CD8+ T cell responses at post-boost memory time points. Therefore, compared to standard regimens, a 16-week interval has a favorable impact on the B cell compartment but minimally affects T cell immunity.

Validating a Proteomic Signature of Severe COVID-19

OBJECTIVES: COVID-19 is a heterogenous disease. Biomarker-based approaches may identify patients at risk for severe disease, who may be more likely to benefit from specific therapies. Our objective was to identify and validate a plasma protein signature for severe COVID-19. DESIGN: Prospective observational cohort study. SETTING: Two hospitals in the United States. PATIENTS: One hundred sixty-seven hospitalized adults with COVID-19. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: We measured 713 plasma proteins in 167 hospitalized patients with COVID-19 using a high-throughput platform. We classified patients as nonsevere versus severe COVID-19, defined as the need for high-flow nasal cannula, mechanical ventilation, extracorporeal membrane oxygenation, or death, at study entry and in 7-day intervals thereafter. We compared proteins measured at baseline between these two groups by logistic regression adjusting for age, sex, symptom duration, and comorbidities. We used lead proteins from dysregulated pathways as inputs for elastic net logistic regression to identify a parsimonious signature of severe disease and validated this signature in an external COVID-19 dataset. We tested whether the association between corticosteroid use and mortality varied by protein signature. One hundred ninety-four proteins were associated with severe COVID-19 at the time of hospital admission. Pathway analysis identified multiple pathways associated with inflammatory response and tissue repair programs. Elastic net logistic regression yielded a 14-protein signature that discriminated 90-day mortality in an external cohort with an area under the receiver-operator characteristic curve of 0.92 (95% CI, 0.88–0.95). Classifying patients based on the predicted risk from the signature identified a heterogeneous response to treatment with corticosteroids (p = 0.006). CONCLUSIONS: Inpatients with COVID-19 express heterogeneous patterns of plasma proteins. We propose a 14-protein signature of disease severity that may have value in developing precision medicine approaches for COVID-19 pneumonia.

A boost with SARS-CoV-2 BNT162b2 mRNA vaccine elicits strong humoral responses independently of the interval between the first two doses.

Due to the recrudescence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections worldwide, mainly caused by the Omicron variant of concern (VOC) and its sub-lineages, several jurisdictions are administering an mRNA vaccine boost. Here, we analyze humoral responses induced after the second and third doses of an mRNA vaccine in naive and previously infected donors who received their second dose with an extended 16-week interval. We observe that the extended interval elicits robust humoral responses against VOCs, but this response is significantly diminished 4 months after the second dose. Administering a boost to these individuals brings back the humoral responses to the same levels obtained after the extended second dose. Interestingly, we observe that administering a boost to individuals that initially received a short 3- to 4-week regimen elicits humoral responses similar to those observed in the long interval regimen. Nevertheless, humoral responses elicited by the boost in naive individuals do not reach those present in previously infected vaccinated individuals.

SARS-CoV-2 infections elicit higher levels of original antigenic sin antibodies compared with SARS-CoV-2 mRNA vaccinations.

It is important to determine if severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and SARS-CoV-2 mRNA vaccinations elicit different types of antibodies. Here, we characterize the magnitude and specificity of SARS-CoV-2 spike-reactive antibodies from 10 acutely infected health care workers with no prior SARS-CoV-2 exposure history and 23 participants who received SARS-CoV-2 mRNA vaccines. We found that infection and primary mRNA vaccination elicit S1- and S2-reactive antibodies, while secondary vaccination boosts mostly S1 antibodies. Using absorption assays, we found that SARS-CoV-2 infections elicit a large proportion of original antigenic sin-like antibodies that bind efficiently to the spike of common seasonal human coronaviruses but poorly to the spike of SARS-CoV-2. In converse, vaccination modestly boosts antibodies reactive to the spike of common seasonal human coronaviruses, and these antibodies cross-react more efficiently to the spike of SARS-CoV-2. Our data indicate that SARS-CoV-2 infections and mRNA vaccinations elicit fundamentally different antibody responses.

A boost with SARS-CoV-2 BNT162b2 mRNA vaccine elicits strong humoral responses independently of the interval between the first two doses

Due to the recrudescence of SARS-CoV-2 infections worldwide, mainly caused by Omicron variant of concern (VOC) and its sub-lineages, several jurisdictions are administering a mRNA vaccine boost. Here, we analyze humoral responses induced after the second and third doses of mRNA vaccine in naïve and previously-infected donors who received their second dose with an extended 16-week interval. We observe that the extended interval elicits robust humoral responses against VOCs, but this response is significantly diminished 4 months after the second dose. Administering a boost to these individuals brings back the humoral responses to the same levels obtained after the extended second dose. Interestingly, we observe that administering a boost to individuals that initially received a short 3-4 weeks regimen elicits humoral responses similar to those observed in the long interval regimen. Nevertheless, humoral responses elicited by the boost in naïve individuals do not reach those present in previously-infected vaccinated individuals. Graphical In this study, Tauzin et al. report that the third dose of SARS-CoV-2 mRNA vaccine elicits strong humoral responses against VOCs in naïve individuals, regardless of the interval between the first two doses. However, these responses remain lower than those induced by hybrid immunity.

Antigenic and virological properties of an H3N2 variant that continues to dominate the 2021-22 Northern Hemisphere influenza season.

Influenza viruses circulated at very low levels during the beginning of the COVID-19 pandemic, and population immunity against these viruses is low. An H3N2 strain (3C.2a1b.2a2) with a hemagglutinin (HA) that has several substitutions relative to the 2021-22 H3N2 vaccine strain is dominating the 2021-22 Northern Hemisphere influenza season. Here, we show that one of these substitutions eliminates a key glycosylation site on HA and alters sialic acid binding. Using glycan array profiling, we show that the 3C.2a1b.2a2 H3 maintains binding to an extended biantennary sialoside and replicates to high titers in human airway cells. We find that antibodies elicited by the 2021-22 Northern Hemisphere influenza vaccine poorly neutralize the 3C.2a1b.2a2 H3N2 strain. Together, these data indicate that 3C.2a1b.2a2 H3N2 viruses efficiently replicate in human cells and escape vaccine-elicited antibodies.

Signaling Through FcγRIIA and the C5a-C5aR Pathway Mediate Platelet Hyperactivation in COVID-19.

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect.

Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine.

We examined antibody and memory B cell responses longitudinally for ∼9-10 months after primary 2-dose SARS-CoV-2 mRNA vaccination and 3 months after a 3rd dose. Antibody decay stabilized between 6 and 9 months, and antibody quality continued to improve for at least 9 months after 2-dose vaccination. Spike- and RBD-specific memory B cells remained durable over time, and 40%-50% of RBD-specific memory B cells simultaneously bound the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells were efficiently reactivated by a 3rd dose of wild-type vaccine and correlated with the corresponding increase in neutralizing antibody titers. In contrast, pre-3rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit the added protection afforded by repeat short interval boosting. These data provide insight into the quantity and quality of mRNA-vaccine-induced immunity over time through 3 or more antigen exposures.

Germinal center responses to SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals.

Vaccine-mediated immunity often relies on the generation of protective antibodies and memory B cells, which commonly stem from germinal center (GC) reactions. An in-depth comparison of the GC responses elicited by SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals has not yet been performed due to the challenge of directly probing human lymph nodes. Herein, through a fine-needle aspiration-based approach, we profiled the immune responses to SARS-CoV-2 mRNA vaccines in lymph nodes of healthy individuals and kidney transplant recipients (KTXs). We found that, unlike healthy subjects, KTXs presented deeply blunted SARS-CoV-2-specific GC B cell responses coupled with severely hindered T follicular helper cell, SARS-CoV-2 receptor binding domain-specific memory B cell, and neutralizing antibody responses. KTXs also displayed reduced SARS-CoV-2-specific CD4 and CD8 T cell frequencies. Broadly, these data indicate impaired GC-derived immunity in immunocompromised individuals and suggest a GC origin for certain humoral and memory B cell responses following mRNA vaccination.

Cellular and humoral immune responses following SARS-CoV-2 mRNA vaccination in patients with multiple sclerosis on anti-CD20 therapy.

SARS-CoV-2 messenger RNA vaccination in healthy individuals generates immune protection against COVID-19. However, little is known about SARS-CoV-2 mRNA vaccine-induced responses in immunosuppressed patients. We investigated induction of antigen-specific antibody, B cell and T cell responses longitudinally in patients with multiple sclerosis (MS) on anti-CD20 antibody monotherapy (n = 20) compared with healthy controls (n = 10) after BNT162b2 or mRNA-1273 mRNA vaccination. Treatment with anti-CD20 monoclonal antibody (aCD20) significantly reduced spike-specific and receptor-binding domain (RBD)-specific antibody and memory B cell responses in most patients, an effect ameliorated with longer duration from last aCD20 treatment and extent of B cell reconstitution. By contrast, all patients with MS treated with aCD20 generated antigen-specific CD4 and CD8 T cell responses after vaccination. Treatment with aCD20 skewed responses, compromising circulating follicular helper T (TFH) cell responses and augmenting CD8 T cell induction, while preserving type 1 helper T (TH1) cell priming. Patients with MS treated with aCD20 lacking anti-RBD IgG had the most severe defect in circulating TFH responses and more robust CD8 T cell responses. These data define the nature of the SARS-CoV-2 vaccine-induced immune landscape in aCD20-treated patients and provide insights into coordinated mRNA vaccine-induced immune responses in humans. Our findings have implications for clinical decision-making and public health policy for immunosuppressed patients including those treated with aCD20.

Multisystem Inflammation and Organ Dysfunction After BNT162b2 Messenger RNA Coronavirus Disease 2019 Vaccination.

The U.S. Food and Drug Administration has to date granted approval or emergency use authorization to three vaccines against severe acute respiratory syndrome coronavirus 2 and coronavirus disease 2019. In clinical trials and real-use observational studies, the Pfizer-BioNTech BNT162b2 messenger RNA coronavirus disease 2019 vaccine, as well as the Moderna mRNA-1273 messenger RNA coronavirus disease 2019 vaccine, have demonstrated high efficacy and few adverse events. CASE SUMMARY: A 20-year-old male college student in good health developed tinnitus and hematuria shortly after vaccination and progressed swiftly to a syndrome of: systemic inflammation; acute kidney injury requiring hemodialysis; acute, bilateral, complete sensorineural hearing loss; radiographic evidence of acute multifocal ischemic strokes; pericardial effusion complicated by tamponade physiology requiring pericardial evacuation; pleural effusions requiring evacuation; and systemic capillary leak. An extensive clinical and research investigation, including cytokine analysis, whole blood cytometry by time of flight, and whole exome sequencing, did not reveal a definitive explanatory mechanism. CONCLUSION: While the overall safety profile of the BNT162b2 coronavirus disease 2019 vaccine remains excellent for the general population, rare serious events have been reported. In this report, we describe a case of multisystem inflammation and organ dysfunction of unknown mechanism beginning shortly after administration of the first dose of BNT162b2 coronavirus disease 2019 vaccine in a previously healthy recipient.

SARS-CoV-2 Seroprevalence Among Parturient Women

Limited data are available for pregnant women affected by SARS-CoV-2. Serological tests are critically important to determine exposure and immunity to SARS-CoV-2 within both individuals and populations. We completed SARS-CoV-2 serological testing of 1,293 parturient women at two centers in Philadelphia from April 4 to June 3, 2020. We tested 834 pre-pandemic samples collected in 2019 and 15 samples from COVID-19 recovered donors to validate our assay, which has a ~1% false positive rate. We found 80/1,293 (6.2%) of parturient women possessed IgG and/or IgM SARS-CoV-2-specific antibodies. We found race/ethnicity differences in seroprevalence rates, with higher rates in Black/non-Hispanic and Hispanic/Latino women. Of the 72 seropositive women who also received nasopharyngeal polymerase chain reaction testing during pregnancy, 46 (64%) were positive. Continued serologic surveillance among pregnant women may inform perinatal clinical practices and can potentially be used to estimate seroprevalence within the community.

mRNA vaccines induce durable immune memory to SARS-CoV-2 and variants of concern.

The durability of immune memory after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA (mRNA) vaccination remains unclear. In this study, we longitudinally profiled vaccine responses in SARS-CoV-2­naïve and ­recovered individuals for 6 months after vaccination. Antibodies declined from peak levels but remained detectable in most subjects at 6 months. By contrast, mRNA vaccines generated functional memory B cells that increased from 3 to 6 months postvaccination, with the majority of these cells cross-binding the Alpha, Beta, and Delta variants. mRNA vaccination further induced antigen-specific CD4+ and CD8+ T cells, and early CD4+ T cell responses correlated with long-term humoral immunity. Recall responses to vaccination in individuals with preexisting immunity primarily increased antibody levels without substantially altering antibody decay rates. Together, these findings demonstrate robust cellular immune memory to SARS-CoV-2 and its variants for at least 6 months after mRNA vaccination.

Rapid induction of antigen-specific CD4+ T cells is associated with coordinated humoral and cellular immunity to SARS-CoV-2 mRNA vaccination.

SARS-CoV-2 mRNA vaccines have shown remarkable clinical efficacy, but questions remain about the nature and kinetics of T cell priming. We performed longitudinal antigen-specific T cell analyses on healthy SARS-CoV-2-naive and recovered individuals prior to and following mRNA prime and boost vaccination. Vaccination induced rapid antigen-specific CD4+ T cell responses in naive subjects after the first dose, whereas CD8+ T cell responses developed gradually and were variable in magnitude. Vaccine-induced Th1 and Tfh cell responses following the first dose correlated with post-boost CD8+ T cells and neutralizing antibodies, respectively. Integrated analysis revealed coordinated immune responses with distinct trajectories in SARS-CoV-2-naive and recovered individuals. Last, whereas booster vaccination improved T cell responses in SARS-CoV-2-naive subjects, the second dose had little effect in SARS-CoV-2-recovered individuals. These findings highlight the role of rapidly primed CD4+ T cells in coordinating responses to the second vaccine dose in SARS-CoV-2-naive individuals.

Distinct antibody and memory B cell responses in SARS-CoV-2 naïve and recovered individuals following mRNA vaccination.

Novel mRNA vaccines for SARS-CoV-2 have been authorized for emergency use. Despite their efficacy in clinical trials, data on mRNA vaccine-induced immune responses are mostly limited to serological analyses. Here, we interrogated antibody and antigen-specific memory B cells over time in 33 SARS-CoV-2 naïve and 11 SARS-CoV-2 recovered subjects. SARS-CoV-2 naïve individuals required both vaccine doses for optimal increases in antibodies, particularly for neutralizing titers against the B.1.351 variant. Memory B cells specific for full-length spike protein and the spike receptor binding domain (RBD) were also efficiently primed by mRNA vaccination and detectable in all SARS-CoV-2 naive subjects after the second vaccine dose, though the memory B cell response declined slightly with age. In SARS-CoV-2 recovered individuals, antibody and memory B cell responses were significantly boosted after the first vaccine dose; however, there was no increase in circulating antibodies, neutralizing titers, or antigen-specific memory B cells after the second dose. This robust boosting after the first vaccine dose strongly correlated with levels of pre-existing memory B cells in recovered individuals, identifying a key role for memory B cells in mounting recall responses to SARS-CoV-2 antigens. Together, our data demonstrated robust serological and cellular priming by mRNA vaccines and revealed distinct responses based on prior SARS-CoV-2 exposure, whereby COVID-19 recovered subjects may only require a single vaccine dose to achieve peak antibody and memory B cell responses. These findings also highlight the utility of defining cellular responses in addition to serologies and may inform SARS-CoV-2 vaccine distribution in a resource-limited setting.

CD8+ T cells contribute to survival in patients with COVID-19 and hematologic cancer.

Patients with cancer have high mortality from coronavirus disease 2019 (COVID-19), and the immune parameters that dictate clinical outcomes remain unknown. In a cohort of 100 patients with cancer who were hospitalized for COVID-19, patients with hematologic cancer had higher mortality relative to patients with solid cancer. In two additional cohorts, flow cytometric and serologic analyses demonstrated that patients with solid cancer and patients without cancer had a similar immune phenotype during acute COVID-19, whereas patients with hematologic cancer had impairment of B cells and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody responses. Despite the impaired humoral immunity and high mortality in patients with hematologic cancer who also have COVID-19, those with a greater number of CD8 T cells had improved survival, including those treated with anti-CD20 therapy. Furthermore, 77% of patients with hematologic cancer had detectable SARS-CoV-2-specific T cell responses. Thus, CD8 T cells might influence recovery from COVID-19 when humoral immunity is deficient. These observations suggest that CD8 T cell responses to vaccination might provide protection in patients with hematologic cancer even in the setting of limited humoral responses.

Seasonal human coronavirus antibodies are boosted upon SARS-CoV-2 infection but not associated with protection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread within the human population. Although SARS-CoV-2 is a novel coronavirus, most humans had been previously exposed to other antigenically distinct common seasonal human coronaviruses (hCoVs) before the coronavirus disease 2019 (COVID-19) pandemic. Here, we quantified levels of SARS-CoV-2-reactive antibodies and hCoV-reactive antibodies in serum samples collected from 431 humans before the COVID-19 pandemic. We then quantified pre-pandemic antibody levels in serum from a separate cohort of 251 individuals who became PCR-confirmed infected with SARS-CoV-2. Finally, we longitudinally measured hCoV and SARS-CoV-2 antibodies in the serum of hospitalized COVID-19 patients. Our studies indicate that most individuals possessed hCoV-reactive antibodies before the COVID-19 pandemic. We determined that ∼20% of these individuals possessed non-neutralizing antibodies that cross-reacted with SARS-CoV-2 spike and nucleocapsid proteins. These antibodies were not associated with protection against SARS-CoV-2 infections or hospitalizations, but they were boosted upon SARS-CoV-2 infection.

Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications.

Coronavirus disease 2019 (COVID-19) is currently a global pandemic, but human immune responses to the virus remain poorly understood. We used high-dimensional cytometry to analyze 125 COVID-19 patients and compare them with recovered and healthy individuals. Integrated analysis of ~200 immune and ~50 clinical features revealed activation of T cell and B cell subsets in a proportion of patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses reaching >30% of circulating B cells. However, another subgroup had lymphocyte activation comparable with that in uninfected individuals. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. Our analyses identified three immunotypes associated with poor clinical trajectories versus improving health. These immunotypes may have implications for the design of therapeutics and vaccines for COVID-19.