Adaptive immune responses to SARS-CoV-2 persist in the pharyngeal lymphoid tissue of children.

Most studies of adaptive immunity to SARS-CoV-2 infection focus on peripheral blood, which may not fully reflect immune responses at the site of infection. Using samples from 110 children undergoing tonsillectomy and adenoidectomy during the COVID-19 pandemic, we identified 24 samples with evidence of previous SARS-CoV-2 infection, including neutralizing antibodies in serum and SARS-CoV-2-specific germinal center and memory B cells in the tonsils and adenoids. Single-cell B cell receptor (BCR) sequencing indicated virus-specific BCRs were class-switched and somatically hypermutated, with overlapping clones in the two tissues. Expanded T cell clonotypes were found in tonsils, adenoids and blood post-COVID-19, some with CDR3 sequences identical to previously reported SARS-CoV-2-reactive T cell receptors (TCRs). Pharyngeal tissues from COVID-19-convalescent children showed persistent expansion of germinal center and antiviral lymphocyte populations associated with interferon (IFN)-γ-type responses, particularly in the adenoids, and viral RNA in both tissues. Our results provide evidence for persistent tissue-specific immunity to SARS-CoV-2 in the upper respiratory tract of children after infection.

Pro-regenerative biomaterials recruit immunoregulatory dendritic cells after traumatic injury.

During wound healing and surgical implantation, the body establishes a delicate balance between immune activation to fight off infection and clear debris and immune tolerance to control reactivity against self-tissue. Nonetheless, how such a balance is achieved is not well understood. Here we describe that pro-regenerative biomaterials for muscle injury treatment promote the proliferation of a BATF3-dependent CD103+XCR1+CD206+CD301b+ dendritic cell population associated with cross-presentation and self-tolerance. Upregulation of E-cadherin, the ligand for CD103, and XCL-1 in injured tissue suggests a mechanism for cell recruitment to trauma. Muscle injury recruited natural killer cells that produced Xcl1 when stimulated with fragmented extracellular matrix. Without cross-presenting cells, T-cell activation increases, pro-regenerative macrophage polarization decreases and there are alterations in myogenesis, adipogenesis, fibrosis and increased muscle calcification. These results, previously observed in cancer progression, suggest a fundamental mechanism of immune regulation in trauma and material implantation with implications for both short- and long-term injury recovery.

Development of a High-Color Flow Cytometry Panel for Immunologic Analysis of Tissue Injury and Reconstruction in a Rat Model.

The rat model is an important resource in biomedical research due to its similarities to the human immune system and its use for functional studies. However, because of the preponderance of mouse models in foundational and mechanistic immunological studies, there is a relative lack of diverse, commercially available flow cytometry antibodies for immunological profiling in the rat model. Available antibodies are often conjugated to common fluorophores with similar peak emission wavelengths, making them hard to distinguish on conventional flow cytometers and restricting more comprehensive immune analysis. This can become a limitation when designing immunological studies in rat injury models to investigate the immune response to tissue injury. In addition, this lack of available antibodies limits the number of studies that can be done on the immune populations in lymphoid organs in other research areas. To address this critical unmet need, we designed a spectral flow cytometry panel for rat models. Spectral cytometry distinguishes between different fluorophores by capturing their full emission spectra instead of their peak emission wavelengths. This flow cytometry panel includes 24 distinct immune cell markers to analyze the innate and adaptive immune response. Importantly, this panel identifies different immune phenotypes, including tolerogenic, Type 1, and Type 2 immune responses. We show that this panel can identify unique immune populations and phenotypes in a rat muscle trauma model. We further validated that the panel can identify distinct adaptive and innate immune populations and their unique phenotypes in lymphoid organs. This panel expands the scope of previous rat panels providing a tool for scientists to examine the immune system in homeostasis and injury while pairing mechanistic immunological studies with functional studies.

SARS-CoV-2 Cross-Reactivity in Prepandemic Serum from Rural Malaria-Infected Persons, Cambodia.

Inhabitants of the Greater Mekong Subregion in Cambodia are exposed to pathogens that might influence serologic cross-reactivity with severe acute respiratory syndrome coronavirus 2. A prepandemic serosurvey of 528 malaria-infected persons demonstrated higher-than-expected positivity of nonneutralizing IgG to spike and receptor-binding domain antigens. These findings could affect interpretation of large-scale serosurveys.

Development of a predictive algorithm for patient survival after traumatic injury using a five analyte blood panel.

Severe trauma can induce systemic inflammation but also immunosuppression, which makes understanding the immune response of trauma patients critical for therapeutic development and treatment approaches. By evaluating the levels of 59 proteins in the plasma of 50 healthy volunteers and 1000 trauma patients across five trauma centers in the United States, we identified 6 novel changes in immune proteins after traumatic injury and further new variations by sex, age, trauma type, comorbidities, and developed a new equation for prediction of patient survival. Blood was collected at the time of arrival at Level 1 trauma centers and patients were stratified based on trauma level, tissues injured, and injury types. Trauma patients had significantly upregulated proteins associated with immune activation (IL-23, MIP-5), immunosuppression (IL-10) and pleiotropic cytokines (IL-29, IL-6). A high ratio of IL-29 to IL-10 was identified as a new predictor of survival in less severe patients with ROC area of 0.933. Combining machine learning with statistical modeling we developed an equation ("VIPER") that could predict survival with ROC 0.966 in less severe patients and 0.8873 for all patients from a five analyte panel (IL-6, VEGF-A, IL-21, IL-29, and IL-10). Furthermore, we also identified three increased proteins (MIF, TRAIL, IL-29) and three decreased proteins (IL-7, TPO, IL-8) that were the most important in distinguishing a trauma blood profile. Biologic sex altered phenotype with IL-8 and MIF being lower in healthy women, but higher in female trauma patients when compared to male counterparts. This work identifies new responses to injury that may influence systemic immune dysfunction, serving as targets for therapeutics and immediate clinical benefit in identifying at-risk patients.

Immunomodulatory contribution of mast cells to the regenerative biomaterial microenvironment.

Bioactive immunomodulatory biomaterials have shown promise for influencing the immune response to promote tissue repair and regeneration. Macrophages and T cells have been associated with this response; however, other immune cell types have been traditionally overlooked. In this study, we investigated the role of mast cells in the regulation of the immune response to decellularized biomaterial scaffolds using a subcutaneous implant model. In mast cell-deficient mice, there was dysregulation of the expected M1 to M2 macrophage transition typically induced by the biomaterial scaffold. Polarization progression deviated in a sex-specific manner with an early transition to an M2 profile in female mice, while the male response was unable to properly transition past a pro-inflammatory M1 state. Both were reversed with adoptive mast cell transfer. Further investigation of the later-stage immune response in male mice determined a greater sustained pro-inflammatory gene expression profile, including the IL-1 cytokine family, IL-6, alarmins, and chemokines. These results highlight mast cells as another important cell type that influences the immune response to pro-regenerative biomaterials.

Examining multi-level immune response to determine prevalence of COVID-19 in pediatric tonsillectomy.

OBJECTIVE: To determine the prevalence of COVID-19 in a cohort of children undergoing tonsillectomy through assessment of B cell immune responses to SARS-CoV-2 in both peripheral blood and tonsil tissue. METHODS: In this cohort study at a tertiary pediatric hospital (Children's National Hospital) in Washington, DC, we recruited 100 children undergoing tonsillectomy from late September 2020 to January 2021. Serum, peripheral blood cells, and tonsil tissue were collected and examined for immune reactivity to SARS-CoV-2. Parent-reported clinical histories were compared to antibody and B-cell responses. RESULTS: Among 100 children undergoing tonsillectomy, 19% had evidence of immune responses to SARS-CoV-2 (CoV2+), indicating prior COVID-19. In all seropositive participants, we detected SARS-CoV-2 specific B cells in both peripheral blood mononuclear cells and tonsils, providing evidence for tissue-specific immunity in these children. Of the 19, 63% reported no known history of COVID-19, and an additional 3 were asymptomatic or unaware of an acute infection when detected on pre-surgery screen. Hispanic children represented 74% of CoV2+ subjects compared to 37% of the full cohort. 100% of CoV2+ children lived in a zip code with poverty level >10%. CONCLUSIONS: Nearly one-fifth of children undergoing tonsillectomy at an urban U.S. hospital had evidence of prior COVID-19 during the early pandemic, with the majority unaware of prior infection. Our results underscore the ethnic and socio-economic disparities of COVID-19. We found concordant evidence of humoral immune responses in children in both blood and tonsil tissue, providing evidence of local immune responses in the upper respiratory tract. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:1993-1999, 2023.

Dynamics of SARS-CoV-2 Seroprevalence in a Large US population Over a Period of 12 Months.

Due to a combination of asymptomatic or undiagnosed infections, the proportion of the United States population infected with SARS-CoV-2 was unclear from the beginning of the pandemic. We previously established a platform to screen for SARS-CoV-2 positivity across a representative proportion of the US population, from which we reported that almost 17 million Americans were estimated to have had undocumented infections in the Spring of 2020. Since then, vaccine rollout and prevalence of different SARS-CoV-2 variants have further altered seropositivity trends within the United States population. To explore the longitudinal impacts of the pandemic and vaccine responses on seropositivity, we re-enrolled participants from our baseline study in a 6- and 12- month follow-up study to develop a longitudinal antibody profile capable of representing seropositivity within the United States during a critical period just prior to and during the initiation of vaccine rollout. Initial measurements showed that, since July 2020, seropositivity elevated within this population from 4.8% at baseline to 36.2% and 89.3% at 6 and 12 months, respectively. We also evaluated nucleocapsid seropositivity and compared to spike seropositivity to identify trends in infection versus vaccination relative to baseline. These data serve as a window into a critical timeframe within the COVID-19 pandemic response and serve as a resource that could be used in subsequent respiratory illness outbreaks.

Characterization of the antispike IgG immune response to COVID-19 vaccines in people with a wide variety of immunodeficiencies.

Research on coronavirus disease 2019 vaccination in immune-deficient/disordered people (IDP) has focused on cancer and organ transplantation populations. In a prospective cohort of 195 IDP and 35 healthy volunteers (HV), antispike immunoglobulin G (IgG) was detected in 88% of IDP after dose 2, increasing to 93% by 6 months after dose 3. Despite high seroconversion, median IgG levels for IDP never surpassed one-third that of HV. IgG binding to Omicron BA.1 was lowest among variants. Angiotensin-converting enzyme 2 pseudo-neutralization only modestly correlated with antispike IgG concentration. IgG levels were not significantly altered by receipt of different messenger RNA-based vaccines, immunomodulating treatments, and prior severe acute respiratory syndrome coronavirus 2 infections. While our data show that three doses of coronavirus disease 2019 vaccinations induce antispike IgG in most IDP, additional doses are needed to increase protection. Because of the notably reduced IgG response to Omicron BA.1, the efficacy of additional vaccinations, including bivalent vaccines, should be studied in this population.

Robust, persistent adaptive immune responses to SARS-CoV-2 in the oropharyngeal lymphoid tissue of children.

SARS-CoV-2 infection triggers adaptive immune responses from both T and B cells. However, most studies focus on peripheral blood, which may not fully reflect immune responses in lymphoid tissues at the site of infection. To evaluate both local and systemic adaptive immune responses to SARS-CoV-2, we collected peripheral blood, tonsils, and adenoids from 110 children undergoing tonsillectomy/adenoidectomy during the COVID-19 pandemic and found 24 with evidence of prior SARS-CoV-2 infection, including detectable neutralizing antibodies against multiple viral variants. We identified SARS-CoV-2-specific germinal center (GC) and memory B cells; single cell BCR sequencing showed that these virus-specific B cells were class-switched and somatically hypermutated, with overlapping clones in the adenoids and tonsils. Oropharyngeal tissues from COVID-19-convalescent children showed persistent expansion of GC and anti-viral lymphocyte populations associated with an IFN-γ-type response, with particularly prominent changes in the adenoids, as well as evidence of persistent viral RNA in both tonsil and adenoid tissues of many participants. Our results show robust, tissue-specific adaptive immune responses to SARS-CoV-2 in the upper respiratory tract of children weeks to months after acute infection, providing evidence of persistent localized immunity to this respiratory virus.

Mobilizing Endogenous Repair Through Understanding Immune Reaction With Biomaterials.

With few exceptions, humans are incapable of fully recovering from severe physical trauma. Due to these limitations, the field of regenerative medicine seeks to find clinically viable ways to repair permanently damaged tissue. There are two main approaches to regenerative medicine: promoting endogenous repair of the wound, or transplanting a material to replace the injured tissue. In recent years, these two methods have fused with the development of biomaterials that act as a scaffold and mobilize the body's natural healing capabilities. This process involves not only promoting stem cell behavior, but by also inducing activity of the immune system. Through understanding the immune interactions with biomaterials, we can understand how the immune system participates in regeneration and wound healing. In this review, we will focus on biomaterials that promote endogenous tissue repair, with discussion on their interactions with the immune system.

Pre-pandemic SARS-CoV-2 serological reactivity in rural malaria-experienced Cambodians.

Greater Mekong inhabitants are exposed to pathogens, zoonotic and otherwise, that may influence SARS-CoV-2 seroreactivity. A pre-pandemic (2005 to 2011) serosurvey of from 528 malaria-experienced Cambodians demonstrated higher-than-expected (up to 13.8 %) positivity of non-neutralizing IgG to SARS-CoV-2 spike and RBD antigens. These findings have implications for interpreting large-scale serosurveys. ARTICLE SUMMARY LINE: In the pre-COVID19 pandemic years of 2005 to 2011, malaria experienced Cambodians from rural settings had higher-than-expected seroreactivity to SARS-CoV-2 spike and receptor binding domain proteins.

SARS-CoV-2 Seroprevalence and Drug Use in Trauma Patients from Six Sites in the United States.

In comparison to the general patient population, trauma patients show higher level detections of bloodborne infectious diseases, such as Hepatitis and Human Immunodeficiency Virus. In comparison to bloodborne pathogens, the prevalence of respiratory infections such as SARS-CoV-2 and how that relates with other variables, such as drug usage and trauma type, is currently unknown in trauma populations. Here, we evaluated SARS-CoV-2 seropositivity and antibody isotype profile in 2,542 trauma patients from six Level-1 trauma centers between April and October of 2020 during the first wave of the COVID-19 pandemic. We found that the seroprevalence in trauma victims 18-44 years old (9.79%, 95% confidence interval/CI: 8.33 - 11.47) was much higher in comparison to older patients (45-69 years old: 6.03%, 4.59-5.88; 70+ years old: 4.33%, 2.54 - 7.20). Black/African American (9.54%, 7.77 - 11.65) and Hispanic/Latino patients (14.95%, 11.80 - 18.75) also had higher seroprevalence in comparison, respectively, to White (5.72%, 4.62 - 7.05) and Non-Latino patients (6.55%, 5.57 - 7.69). More than half (55.54%) of those tested for drug toxicology had at least one drug present in their system. Those that tested positive for narcotics or sedatives had a significant negative correlation with seropositivity, while those on anti-depressants trended positive. These findings represent an important consideration for both the patients and first responders that treat trauma patients facing potential risk of respiratory infectious diseases like SARS-CoV-2.

Undiagnosed SARS-CoV-2 seropositivity during the first 6 months of the COVID-19 pandemic in the United States.

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates in the United States and elsewhere. To address this, we analyzed seropositivity in 9089 adults in the United States who had not been diagnosed previously with COVID-19. Individuals with characteristics that reflected the U.S. population (n = 27,716) were selected by quota sampling from 462,949 volunteers. Enrolled participants (n = 11,382) provided medical, geographic, demographic, and socioeconomic information and dried blood samples. Survey questions coincident with the Behavioral Risk Factor Surveillance System survey, a large probability-based national survey, were used to adjust for selection bias. Most blood samples (88.7%) were collected between 10 May and 31 July 2020 and were processed using ELISA to measure seropositivity (IgG and IgM antibodies against SARS-CoV-2 spike protein and the spike protein receptor binding domain). The overall weighted undiagnosed seropositivity estimate was 4.6% (95% CI, 2.6 to 6.5%), with race, age, sex, ethnicity, and urban/rural subgroup estimates ranging from 1.1% to 14.2%. The highest seropositivity estimates were in African American participants; younger, female, and Hispanic participants; and residents of urban centers. These data indicate that there were 4.8 undiagnosed SARS-CoV-2 infections for every diagnosed case of COVID-19, and an estimated 16.8 million infections were undiagnosed by mid-July 2020 in the United States.

Mapping a Pandemic: SARS-CoV-2 Seropositivity in the United States.

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates. To address this, we analyzed seropositivity in US adults who have not previously been diagnosed with COVID-19. Individuals with characteristics that reflect the US population (n = 11,382) and who had not previously been diagnosed with COVID-19 were selected by quota sampling from 241,424 volunteers (ClinicalTrials.gov NCT04334954). Enrolled participants provided medical, geographic, demographic, and socioeconomic information and 9,028 blood samples. The majority (88.7%) of samples were collected between May 10th and July 31st, 2020. Samples were analyzed via ELISA for anti-Spike and anti-RBD antibodies. Estimation of seroprevalence was performed by using a weighted analysis to reflect the US population. We detected an undiagnosed seropositivity rate of 4.6% (95% CI: 2.6 - 6.5%). There was distinct regional variability, with heightened seropositivity in locations of early outbreaks. Subgroup analysis demonstrated that the highest estimated undiagnosed seropositivity within groups was detected in younger participants (ages 18-45, 5.9%), females (5.5%), Black/African American (14.2%), Hispanic (6.1%), and Urban residents (5.3%), and lower undiagnosed seropositivity in those with chronic diseases. During the first wave of infection over the spring/summer of 2020 an estimate of 4.6% of adults had a prior undiagnosed SARS-CoV-2 infection. These data indicate that there were 4.8 (95% CI: 2.8-6.8) undiagnosed cases for every diagnosed case of COVID-19 during this same time period in the United States, and an estimated 16.8 million undiagnosed cases by mid-July 2020.